Cationic peg-lipids and methods of use

ABSTRACT

The present invention provides cationic-polymer-lipid conjugates (CPLs) such as distal cationic-poly(ethylene glycol)-lipid conjugates which can be incorporated into conventional and stealth liposomes or other lipid-based formulation for enhancing cellular uptake. The CPLs of the present invention comprise a lipid moiety; a hydrophilic polymer; and a polycationic moiety. Method of increasing intracellular delivery of nucleic acids are also provided.

RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional PatentApplication Ser. No. 60/130,151 filed Apr. 20, 1999, the teachings ofwhich are incorporated herein by reference in their entirety for allpurposes.

FIELD OF THE INVENTION

[0002] This invention relates to cationic lipid conjugates, and moreparticularly, to cationic polymer lipid conjugates and lipid-based drugformulations thereof, containing one or more bioactive agents.

BACKGROUND OF THE INVENTION

[0003] Current vectors for gene delivery and gene therapy are comprisedof viral based and non-viral based systems. Lipid-based non-viralsystems include cationic lipid plasmid DNA complexes. Limitations ofthese systems include large sizes, toxicity and instability of thecomplexes in the serum. Unfortunately, the foregoing drawbacks limit theapplications for these complexes.

[0004] Researchers have devoted tremendous effort to the design of longcirculation stealth liposomes that can be used for systemic delivery(see, Papahadjopoulos, D. et al., Proc. Natl. Acad. Sci. 88:11460-11464(1991); Klibanov, A. L. et al., J. Liposome Res., 2:321 (1992); Woodle,M. C. et al., Biochim. Biophys. Acta., 1113:171 (1992); Torchilin, V. P.et al., In: Stealth Liposomes. Ed. By D. Lasic, F. Martin. CRC Press,Boca Raton, Fla., pp. 51-62 (1995); Allen, T. M. et al., Biochim.Biophys. Acta., 1237:99-108 (1995) and Zalipsky, S. et al., J.Controlled Release, 39:153-161 (1996)). In certain instances, anddepending on the formulation, stealth liposomes are often comparativelyinefficient at facilitating cellular uptake and therefore thetherapeutic efficacy is reduced.

[0005] In general, the molecular mechanism of liposomal longevity invivo can be attributed to steric hindrance resulting from hydrophilicpolymer surface barriers. The hydrophilic polymer barriers prevent orreduce the rate of the adsorption of macromolecules from the blood andsterically inhibit both electrostatic and hydrophobic interactionsbetween liposomes and blood components. Thus, although the longevity ofstealth liposomes has been increased by the insertion of hydrophilicpolymers, the cellular uptake of the stealth liposomes often isinefficient.

[0006] Over the past decade, it has also become clear that liposomalsystems possessing cationic lipids are highly effective transfectionagents in vitro (Felgner, P. L. et al., Nature 337:387-388 (1989);Felgner, P. L. et al., Proceedings of the National Academy of Sciencesof the United States of America 84:7413-7417 (1987)). The addition ofcationic liposomes to plasmid DNA gives rise to large DNA-lipidcomplexes that possess excellent transfection properties in vitro, butwhich are ineffective in vivo due to their rapid clearance from thecirculation by cells of the reticuloendothelial system (RES). The needfor a non-viral lipid-based system capable of systemic delivery of genesto cells led to the recent development of stabilized plasmid-lipidparticles (SPLPs) (Wheeler, J. J. et al., Gene Therapy 6:271-281(1999)). These particles are small (about 70 nm), contain a single copyof a plasmid vector, possess stealth properties resulting from a surfacecoating of poly(ethyleneglycol) (PEG), and protect DNA from degradationby serum nucleases.

[0007] Enhancing intracellular delivery of liposomes and/or theircontents represents one of the major remaining problems in thedevelopment of the next generation of drug delivery systems. In order tooptimize the delivery of drugs (conventional or genetic) in vivo,general methods for increasing the interactions of liposomes with cellsneed to be developed. To date, attempts include the use of specifictargeting information on the liposome surface, such as an antibody (see,Meyer, O. et al., Journal of Biological Chemistry 273:15621-15627(1998); Kao, G. Y. et al., Cancer Gene Therapy 3:250-256 (1996); Hansen,C. B. et al., Biochimica et Biophysica Acta 1239:133-144 (1995)),vitamin—(see, Gabizon, A. et al., Bioconjugate Chemistry 10:289-298(1999); Lee, R. J. et al., Journal of Biological Chemistry 269:3198-3204(1994); Reddy, J. A. et al., Critical Reviews in Therapeutic DrugCarrier Systems 15:587-627 (1998); Holladay, S. R. et al., Biochimica etBiophysica Acta 1426:195-204 (1999); Wang, S. et al., Journal ofControlled Release 53:39-48 (1998)), oligopeptide—(see, Zalipsky, S. etal., Bioconjugate Chemistry 6:705-708 (1995); Zalipsky, S. et al.,Bioconjugate Chemistry 8:111-118 (1997)), or the use of oligosaccharideconstructs specific for a particular membrane protein or receptor.Unfortunately, these methods have not been successful in achieving thisgoal, despite promising in vitro results. While specific targeting ofliposomes to tissues remains an important area of research, otherapproaches may also provide significant improvements in theeffectiveness of liposomal carriers.

[0008] In view of the foregoing, what is needed in the art is alipid-based drug formulation with increased longevity coupled withincreased cellular uptake. The present invention satisfies this andother needs.

SUMMARY OF THE INVENTION

[0009] In certain aspects, the present invention relates to newconjugates that can be incorporated or inserted into stabilized plasmidlipid particles to enhance transfection efficiencies. The conjugates ofthe present invention possess a lipid anchor for anchoring the conjugateinto the bilayer lipid particle, wherein the lipid anchor is attached toa non-immunogenic polymer, such as a PEG moiety, and wherein thenon-immunogenic polymer is, in turn, attached to a polycationic moiety,such as a positively charged moiety. As such, the present inventionprovides a compound of Formula I:

A-W-Y  I

[0010] In Formula I, “A” is a lipid moiety attached to a non-immunogenicpolymer. “W,” in Formula I, is a non-immunogenic polymer, and “Y”, inFormula I, is a polycationic moiety.

[0011] In certain preferred embodiments, the compounds of Formula Icontain groups that give rise to compounds having the general structureof Formula II:

[0012] In Formula II, “A” is a lipid, such as a hydrophobic lipid. InFormula II, “X” is a single bond or a functional group that covalentlyattaches the lipid to at least one ethylene oxide unit, i.e.,(—CH₂—CH₂—O—). In Formula II, “Z” is a single bond or a functional groupthat covalently attaches the at least one ethylene oxide unit to acationic group. In Formula II, “Y” is a polycationic moiety. In FormulaII, the index “n” is an integer ranging in value from about 6 to about160.

[0013] In other aspects, the present invention relates to a lipid-baseddrug formulation comprising:

[0014] (a) a compound having Formula I

A-W-Y  I

[0015] wherein: A, W and Y have been defined;

[0016] (b) a bioactive agent; and

[0017] (c) a second lipid.

[0018] In certain embodiments, the lipid-based drug formulation is inthe form of a liposome, a micelle, a virosome, a lipid-nucleic acidparticle, a nucleic acid aggregate and mixtures thereof. In certainother embodiments, the bioactive agent is a therapeutic nucleic acid orother drugs.

[0019] In yet other aspects, the present invention relates to a methodfor increasing intracellular delivery of a lipid-based drug deliverysystem, comprising: incorporating into the lipid-based drug deliverysystem a compound of Formulae I or II, thereby increasing theintracellular delivery of the lipid-based drug delivery system.

[0020] Additional aspects and advantages of the present invention willbe apparent when read with the following detailed description and theaccompanying drawings.

Definitions

[0021] The term “lipid” refers to a group of organic compounds thatinclude, but are not limited to, esters of fatty acids and arecharacterized by being insoluble in water, but soluble in many organicsolvents. They are usually divided into at least three classes: (1)“simple lipids” which include fats and oils as well as waxes; (2)“compound lipids” which include phospholipids and glycolipids; (3)“derived lipids” such as steroids.

[0022] The term “vesicle-forming lipid” is intended to include anyamphipathic lipid having a hydrophobic moiety and a polar head group,and which by itself can form spontaneously into bilayer vesicles inwater, as exemplified by most phospholipids.

[0023] The term “vesicle-adopting lipid” is intended to include anyamphipathic lipid which is stably incorporated into lipid bilayers incombination with other amphipathic lipids, with its hydrophobic moietyin contact with the interior, hydrophobic region of the bilayermembrane, and its polar head group moiety oriented toward the exterior,polar surface of the membrane. Vesicle-adopting lipids include lipidsthat on their own tend to adopt a non-lamellar phase, yet which arecapable of assuming a bilayer structure in the presence of abilayer-stabilizing component. A typical example is DOPE(dioleoylphosphatidylethanolamine). Bilayer stabilizing componentsinclude, but are not limited to, polyamide oligomers, peptides,proteins, detergents, lipid-derivatives, PEG-lipid derivatives such asPEG coupled to phosphatidylethanolamines, and PEG conjugated toceramides (see, U.S. application Ser. No. 08/485,608, now U.S. Pat. No.5,885,613, which is incorporated herein by reference).

[0024] The term “amphipathic lipid” refers, in part, to any suitablematerial wherein the hydrophobic portion of the lipid material orientsinto a hydrophobic phase, while the hydrophilic portion orients towardthe aqueous phase. Amphipathic lipids are usually the major component ofa lipid vesicle. Hydrophilic characteristics derive from the presence ofpolar or charged groups such as carbohydrates, phosphato, carboxylic,sulfato, amino, sulfbydryl, nitro, hydroxy and other like groups.Hydrophobicity can be conferred by the inclusion of apolar groups thatinclude, but are not limited to, long chain saturated and unsaturatedaliphatic hydrocarbon groups and such groups substituted by one or morearomatic, cycloaliphatic or heterocyclic group(s). Examples ofamphipathic compounds include, but are not limited to, phospholipids,aminolipids and sphingolipids. Representative examples of phospholipidsinclude, but are not limited to, phosphatidylcholine,phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol,phosphatidic acid, palmitoyloleoyl phosphatidylcholine,lysophosphatidylcholine, lysophosphatidylethanolamine,dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine,distearoylphosphatidylcholine or dilinoleoylphosphatidylcholine. Othercompounds lacking in phosphorus, such as sphingolipid, glycosphingolipidfamilies, diacylglycerols and β-acyloxyacids, are also within the groupdesignated as amphipathic lipids. Additionally, the amphipathic lipiddescribed above can be mixed with other lipids including triglyceridesand sterols.

[0025] The term “neutral lipid” refers to any of a number of lipidspecies that exist either in an uncharged or neutral zwitterionic format a selected pH. At physiological pH, such lipids include, for example,diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide,sphingomyelin, cephalin, cholesterol, cerebrosides and diacylglycerols.

[0026] The term “hydrophopic lipid” refers to compounds having apolargroups that include, but are not limited to, long chain saturated andunsaturated aliphatic hydrocarbon groups and such groups optionallysubstituted by one or more aromatic, cycloaliphatic or heterocyclicgroup(s). Suitable examples include, but are not limited to,diacylglycerol, dialkylglycerol, N-N-dialkylamino,1,2-diacyloxy-3-aminopropane and 1,2-dialkyl-3-aminopropane.

[0027] The term “diacylglycerolyl” denotes 2-fatty acyl chains, R¹ andR² having independently between 2 and 30 carbons bonded to the 1- and2-position of glycerol by ester linkages. The acyl groups can besaturated or have varying degrees of unsaturation. Diacylglycerol groupshave the following general formula:

[0028] The term “dialkylglycerolyl” denotes two C₁-C₃₀ alkyl chainsbonded to the 1- and 2-position of glycerol by ether linkages.Dialkylglycerol groups have the following general formula:

[0029] The term “N—N-dialkylamino” denotes

[0030] The term “1,2-diacyloxy-3-aminopropane” denotes 2-fatty acylchains C₁-C₃₀ bonded to the 1- and 2-position of propane by an esterlinkage. The acyl groups can be saturated or have varying degrees ofunsaturation. The 3-position of the propane molecule has a —NH— groupattached. 1,2-diacyloxy-3-aminopropanes have the following generalformula:

[0031] The term “1,2-dialkyl-3-aminopropane” denotes 2-alkyl chains(C₁-C₃₀) bonded to the 1- and 2-position of propane by an ether linkage.The 3-position of the propane molecule has a —NH— group attached.1,2-dialkyl-3-aminopropanes have the following general formula:

[0032] The term “non-cationic lipid” refers to any neutral lipid asdescribed above as well as anionic -lipids. Examples of anionic lipidsinclude, but are not limited to, phosphatidylglycerol, cardiolipin,diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamines, N-succinyl phosphatidylethanolamines,N-glutarylphosphatidylethanolamines, lysophosphatidylglycerols, andother anionic modifying groups joined to neutral lipids.

[0033] The term “cationic lipid” refers to any of a number of lipidspecies that carry a net positive charge at a selected pH, such asphysiological pH. Such lipids include, but are not limited to,N,N-dioleyl-N,N-dimethylammonium chloride (“DODAC”);N-(2,3-dioleyloxy)propyl)-N,N,N-t rimethylammonium chloride (“DOTMA”);N,N-distearyl-N,N-dimethylammonium bromide (“DDAB”);N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (“DOTAP”);3-(N-(N′,N′-dimethylaminoethane)-carbamoyl)cholesterol (“DC-Chol”) andN-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide (“DMRIE”). Additionally, a number of commercial preparations ofcationic lipids are available which can be used in the presentinvention. These include, for example, LIPOFECTIN® (commerciallyavailable cationic liposomes comprising DOTMA and1,2-dioleoyl-sn-3-phosphoethanolamine (“DOPE”), from GIBCO/BRL, GrandIsland, N.Y., USA); LIPOFECTAMINE® (commercially available cationicliposomes comprisingN-(1-(2,3-dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammoniumtrifluoroacetate (“DOSPA”) and (“DOPE”), from GIBCO/BRL); andTRANSFECTAM® (commercially available cationic lipids comprisingdioctadecylamidoglycyl carboxyspermine (“DOGS”) in ethanol from PromegaCorp., Madison, Wis., USA). The following lipids are cationic and have apositive charge at below physiological pH: DODAP, DODMA, DMDMA and thelike.

[0034] The term “fusogenic” refers to the ability of a liposome or otherdrug delivery system to fuse with membranes of a cell. The membranes canbe either the plasma membrane or membranes surrounding organelles, e.g.,endosome, nucleus, etc. Fusogenesis is the fusion of a liposome to sucha membrane.

[0035] The term “dendrimer” includes reference to branched polymers thatpossess multiple generations. In dendrimers, each generation createsmultiple branch points.

[0036] The term “ligand” includes any molecule, compound or device witha reactive functional group and includes lipids, amphipathic lipids,carrier compounds, bioaffinity compounds, biomaterials, biopolymers,biomedical devices, analytically detectable compounds, therapeuticallyactive compounds, enzymes, peptides, proteins, antibodies, immunestimulators, radiolabels, fluorogens, biotin, drugs, haptens, DNA, RNA,polysaccharides, liposomes, virosomes, micelles, immunoglobulins,functional groups, targeting agents, or toxins. The foregoing list isillustrative and not intended to be exhaustive.

[0037] The term “ATTA” or “polyamide” refers to, but is not limited to,compounds disclosed in U.S. patent application Ser. No. 09/218,988,filed Dec. 22, 1998. These compounds include a compound having theformula

[0038] wherein: R is a member selected from the group consisting ofhydrogen, alkyl and acyl; R¹ is a member selected from the groupconsisting of hydrogen and alkyl; or optionally, R and R¹ and thenitrogen to which they are bound form an azido moiety; R² is a member ofthe group selected from hydrogen, optionally substituted alkyl,optionally substituted aryl and a side chain of an amino acid; R³ is amember selected from the group consisting of hydrogen, halogen, hydroxy,alkoxy, mercapto, hydrazino, amino and NR⁴R⁵, wherein R⁴ and R⁵ areindependently hydrogen or alkyl; n is 4 to 80; m is 2 to 6; p is 1 to 4;and q is 0 or 1. It will be apparent to those of skill in the art thatother polyamides can be used in the compounds of the present invention.

[0039] As used herein, the term “alkyl” denotes branched or unbranchedhydrocarbon chains, such as, methyl, ethyl, n-propyl, iso-propyl,n-butyl, sec-butyl, iso-butyl, tertbutyl, octa-decyl and 2-methylpentyl.These groups can be optionally substituted with one or more functionalgroups which are attached commonly to such chains, such as, hydroxyl,bromo, fluoro, chloro, iodo, mercapto or thio, cyano, alkylthio,heterocyclyl, aryl, heteroaryl, carboxyl, carbalkoyl, alkyl, alkenyl,nitro, amino, alkoxyl, amido, and the like to form alkyl groups such astrifluoromethyl, 3-hydroxyhexyl, 2-carboxypropyl, 2-fluoroethyl,carboxymethyl, cyanobutyl and the like.

[0040] The term “alkylene” refers to a divalent alkyl as defined above,such as methylene (—CH₂—), propylene (—CH₂CH₂CH₂—), chloroethylene(—CHClCH₂—), 2-thiobutene (—CH₂CH(SH)CH₂CH₂—),1-bromo-3-hydroxyl-4-methylpentene (—CHBrCH₂CH(OH)CH(CH₃)CH₂—), and thelike.

[0041] The term “alkenyl” denotes branched or unbranched hydrocarbonchains containing one or more carbon-carbon double bonds.

[0042] The term “alkynyl” refers to branched or unbranched hydrocarbonchains containing one or more carbon-carbon triple bonds.

[0043] The term “aryl” denotes a chain of carbon atoms which form atleast one aromatic ring having preferably between about 6-14 carbonatoms, such as phenyl, naphthyl, indenyl, and the like, and which may besubstituted with one or more functional groups which are attachedcommonly to such chains, such as hydroxyl, bromo, fluoro, chloro, iodo,mercapto or thio, cyano, cyanoamido, alkylthio, heterocycle, aryl,heteroaryl, carboxyl, carbalkoyl, alkyl, alkenyl, nitro, amino, alkoxyl,amido, and the like to form aryl groups such as biphenyl, iodobiphenyl,methoxybiphenyl, anthryl, bromophenyl, iodophenyl, chlorophenyl,hydroxyphenyl, methoxyphenyl, formylphenyl, acetylphenyl,trifluoromethylthiophenyl, trifluoromethoxyphenyl, alkylthiophenyl,trialkylamrnmoniumphenyl, amidophenyl, thiazolylphenyl, oxazolylphenyl,imidazolylphenyl, imidazolylmethylphenyl, and the like.

[0044] The term “acyl” denotes the —C(O)R group, wherein R is alkyl oraryl as defined above, such as formyl, acetyl, propionyl, or butyryl.

[0045] The term “alkoxy” denotes —OR—, wherein R is alkyl.

[0046] The term “amido” denotes an amide linkage: —C(O)NR— (wherein R ishydrogen or alkyl).

[0047] The term “amino” denotes an amine linkage: —NR—, wherein R ishydrogen or alkyl or a terminal NH₂.

[0048] The term “carboxyl” denotes the group —C(O)O—, and the term“carbonyl” denotes the group —C(O)—.

[0049] The term “carbonate” indicates the group —OC(O)O—.

[0050] The term “carbamate” denotes the group —NHC(O)O—, and the term“urea” denotes the group —NHC(O)NH—.

[0051] The term “phosphoro” denotes the group —OP(O)(OH)O—.

[0052] The term “basic amino acid” refers to naturally-occurring aminoacids as well as synthetic amino acids and/or or amino acid mimeticshaving a net positive charge at a selected pH, such as physiological pH.This group includes, but is not limited to, lysine, arginine,asparagine, glutamine, histidine and the like.

[0053] The term “phosphorylethanolamino” denotes the group—OP(O)(OH)OCH₂CH₂NH—.

[0054] The term “phosphorylethanolamido” denotes the group—OP(O)(OH)OCH₂CH₂NHC(O)—.

[0055] The term “phospho” denotes a pentavalent phosphorous moiety—P(O)(OH)O—.

[0056] The term “phosphoethanolamino” denotes the group—P(O)(OH)OCH₂CH₂NH—.

[0057] The term “phosphoethanolamido” denotes thegroup-P(O)(OH)OCH₂CH₂NHC(O)—.

[0058] The term “ethylene oxide unit” denotes the group —OCH₂CH₂—.

[0059] The term “CPL” refers to a cationic-polymer-lipid e.g.,cationic-PEG-lipid. Preferred CPLs are compounds of Formulae I and II.

[0060] The term “d-DSPE-CPL-M” is encompassed by the term “CPL1” whichrefers to a DSPE-CPL having one positive charge. The “d-” ind-DSPE-CPL-M indicates that the CPL contains a fluorescent dansyl group.It will be apparent to those of skill in the art that a CPL can besynthesized without the dansyl moiety, and thus the term “DSPE-CPL-M” isencompassed by in the term “CPL1” as defined above.

[0061] The term “d-DSPE-CPL-D” is encompassed by the term “CPL2” whichrefers to DSPE-CPL having two positive charges.

[0062] The term “d-DSPE-CPL-T1” is encompassed by the term “CPL3” whichrefers to DSPE-CPL having three positive charges.

[0063] The term “d-DSPE-CPL-Q1” is encompassed by the term “CPL4a” whichrefers to DSPE-CPL having four positive charges.

[0064] The term “d-DSPE-CPL-Q5,” or, alternatively, DSPE-PEGQuad5, or,alternatively, DSPE-CPL-4, are all encompassed by the term “CPL4 (orCPL4b)” which refer to a DSPE-CPL having four positive charges. Bymodifying the headgroup region, CPLs were synthesized which contained 1(mono, or M), 2 (di, or D), 3 (tri, or T), and 4 (quad, or Q) positivecharges. Various Quad CPLs were synthesized, hence these are numbered Q1through Q5.

[0065] The abbreviations “HBS” refers to Hepes-buffered saline, “Rho-PE”refers to rhodamine-phosphatidylethanolamine, and “LUVs” refers to“large unilamellar vesicles.”

BRIEF DESCRIPTION OF THE DRAWINGS

[0066]FIG. 1 illustrates a structural design of a cationic-polymer lipid(CPL) conjugate.

[0067]FIG. 2 illustrates a synthetic scheme for the preparation ofcationic-PEG-lipid conjugates having varying amount of charged headgroups (a.) Et₃N/CHCl₃; (b.) TFA /CHCl₃; c. Et₃N/CHCl₃ Nα,Nε-di-t-Boc-L-Lysine N-hydroxysuccinide ester.

[0068]FIG. 3 illustrates a CPL incorporated liposome. The largeunilamellar vesicles (LUV) have incorporated different examples of CPLs(CPL1, CPL2, CPL4, and CPL8, respectively).

[0069]FIG. 4 illustrates a distribution of DSPE-CPL-4 between theinner/outer leaflets of a liposomal membrane. CPL-4-LUVs(DSPC/Chol/DSPE-CPL-4, 55:40:5 mole %) were prepared by extrusion methodas described herein. The distribution of outer leaflet CPLs wasquantified by a fluorescamine assay. For the outer leaflet CPLs, thefollowing assay was used. An appropriate amount of CPL-4-LUVs wasdiluted with 1 M Borate buffer (pH 8.5) and cooled in ice-water. 20 μlof 10% Triton X-100 was added to the above sample solution to solubilizethe membrane, and then an additional 20 μl of a cooled fluorescamineethanol solution (10 mg/ml) was added and then measured.

[0070]FIG. 5 illustrates a cellular uptake study of CPL-4 LUVs in BHKcells in PBS-CMG. The controls were LUVs (DSPC/Chol, 60:40) andCPL-4-LUVs (DSPC/Ch/DSPE-CPL-4, 55:40:5) were prepared by extrusion asdescribed herein.

[0071]FIG. 6 illustrates cellular uptake of CPL-4-LUVs in BHK cells inDMEM (with 10% FBS). The control used LUVs (DSPC/Chol, 60:40).CPL-4-LUVs (DSPC/Ch/DSPE-CPL-4, 55:40:5), which were prepared byextrusion as described herein.

[0072]FIG. 7 illustrates a cellular uptake of CPL-liposomes in BHK cellsin PBS-CMG after 4 hr incubation. LUVs (DSPC/Chol, 60:40) and CPL-LUVs(DSPC/Chol/DSPE-CPL, 55:40:5) were prepared by extrusion as describedherein.

[0073]FIG. 8 illustrates the preparation of CPL-LUVs by detergentdialysis. Lipids were codissolved in chloroform at the indicated ratios,following which the solvent was removed by nitrogen gas and high vacuum.The lipid mixture was dissolved in detergent/buffer (OGP in HBS) anddialysed against HBS for 2-3 days. The LUVs, which formed duringdialysis, were then fractionated as shown on Sepharose CL-4B. Panel A:Fractionation of DOPE/DODAC/CPL4[3.4K]/PEGCerC20/Rho-PE(79.5/6/4/10/0.5); Panel B: Fractionation ofDOPE/DODAC/CPL4[1K]/PEGCerC20/Rho-PE (79.5/6/4/10/0.5); and Panel C:Fractionation of DOPE/DODAC/CPL4[3.4K]/PEGCerC20/Rho-PE(71.5/6/12/10/0.5).

[0074]FIG. 9 Panel A illustrates the insertion of DSPE-CPL-Q5 into DOPCLUVs (100 mm). DOPC LUVs (2.5 μmol lipid) were incubated with 0.214 mmolDSPE-CPL-Q5 (total volume 300 μL) at 60° C. for 3 hours, following whichthe sample was applied to a column of Sepharose CL-4B equilibrated inHEPES-buffered saline. 1 mL fractions were collected and assayed fordansyl-labelled CPL and rhodamine-PE as described herein. Panel Billustrates the insertion of DSPE-CPL-Q5 into LUVs (100 nm) composed ofDOPE/DODAC/PEG-Cer-C20 (84/6/10). LUVs (5 μmol lipid) were incubatedwith 0.43 μmol DSPE-CPL-Q5 (total volume 519 μl) at 60° C. for 3 hours,following which the sample was applied to a column of Sepharose CL-4Bequilibrated in HEPES-buffered saline. The elution of free CPL is alsoshown, demonstrating a straightforward method for isolation of theCPL-LUV. 1 mL fractions were collected and assayed for dansyl-labelledCPL and rhodamine-PE as described herein. Panel C illustrates retentionof DSPE-CPL-Q5 in LUVs (100 nm) composed of DOPE/DODAC/PEG-Cer-C20(84/6/10). The main LUV fraction from FIG. 9 Panel B was re-applied to acolumn of Sepharose CL-4B equilibrated in HEPES-buffered saline. 1 mLfractions were collected and assayed for dansyl-labelled CPL andrhodamine-PE as described.

[0075]FIG. 10 illustrates the effect of time and temperature on theinsertion of d-DSPE-CPL-Q1 into DOPE/DODAC/PEG-Cer-C20 LUVs. For each ofthe 3 temperatures, 3 μmol lipid was combined with 0.17 μmol CPL (totalvolume 240 μl). At 1, 3, and 6 hours, 1 μmol of lipid was withdrawn andcooled in ice to halt insertion of CPL. The samples were passed down acolumn of Sepharose CL-4B to remove excess CPL, and assayed for CPLinsertion.

[0076]FIG. 11 illustrates the effect of initial CPL/lipid ratio on finalCPL insertion levels. Initial CPL/lipid molar ratios were 0.011, 0.024,0.047, 0.071, 0.095, and 0.14. Final mol % inserted were 0.8, 1.8, 3.4,5.0, 6.5, and 7.0. Right-hand axis is %-insertion.

[0077]FIG. 12 illustrates the insertion of DSPE-CPL-Q1 and DSPE-CPL-Q5into neutral vesicles. The initial CPL/lipid molar ratio was 0.065 forQ1 (2.5 μmol lipid and 0.21 μmol CPL) and 0.034 for Q5. Samples wereincubated at 60° C. for 3 hours. The DOPC and DOPC/Chol LUVs wereprepared by extrusion, while the others were prepared by detergentdialysis. As described herein, the presence of 4% methanol in the Q5samples appear to account for the higher insertion observed for thissample. Sample compositions were as follows: DOPC/Chol (55/45),DOPC/PEG-Cer-C20 (90/10), DOPC/Chol/PEG-Cer-C20 (45/45/10).

[0078]FIG. 13 illustrates the effect of chain length of PEG-Cer on mol-%CPL inserted. LUVs composed of DOPE/DODAC/PEG-Cer-C20 (84/6/10),DOPE/DODAC/PEG-Cer-C14 (84/6/10), and DOPE/DODAC/PEG-Cer-C8 (79/6/15)were incubated in the presence of between 2-8.6 mol % d-DSPE-CPL-Q1 at60° C. for 3 hrs.

[0079]FIG. 14 illustrates the effect of PEG-Cer-C20 content on insertionof d-DSPE-CPL-Q5. Vesicles composed of DOPC/DODAC/PEGCerC20, with thelatter lipid ranging from 4-10 mol %, were incubated in the presence ofCPL-Q5 (initial CPL/lipid molar ratio=0.071).

[0080]FIG. 15 illustrates the uptake of CPL-LUVs incubated in PBS/CMG onBHK cells. Approximately 105 BHK cells were incubated with 20 nmol ofDOPE/DODAC/PEGCerC20 (84/6/10) LUVs containing (1) no CPL, (2) 8%DSPE-CPL-D, (3) 7% DSPE-CPL-T 1, and (4) 4% DSPE-CPL-Q5. Incubationswere performed at 4° C. and 37° C., the former giving an estimate ofcell binding, and the latter of binding and uptake. By taking thedifference of the two values, an estimate of lipid uptake at 37° C. wasobtained.

[0081]FIG. 16 Panel A illustrates a structure of the CPL₄. Panel Billustrates a protocol for the insertion of CPL₄ into the SPLP system.

[0082]FIG. 17 Panel A illustrates a model for DOPE/DODAC/PEG-Cer-C20LUVs, i.e., a standard liposome containing a PEG-lipid (or “stealth”lipid); Panel B illustrates the same LUVs with CPL₄ (i.e. long chain)inserted. “Long chain” refers to the polymer W being the same length orgreater length than the polymer component of the PEG-lipid. Thus, thecharged group of the CPL1 is immediately exposed to the outsideenvironment; and Panel C illustrates the same LUVs with CPL₄ with ashort chain inserted. A “short chain” CPL, wherein polymer W is shorterthan the corresponding polymer of the PEG-lipid.

[0083]FIG. 18 Panel A illustrates a time-course for the uptake of SPLPsystem () compared to DOPE:DODAC (1:1) liposomes complexed to pLuc (▪)on BHK cells. Lipid concentration was 20 μM. Panel B illustratestransfection efficiencies of 1.5 μg/mL pLuc obtained using the SPLPsystem compared to those obtained using complexes after 4 hour (▪) or 8hour (▪) incubations.

[0084]FIG. 19 illustrates a column profile, following insertion of 3.5mol %_(initial) (3 mol %_(final)) CPL₄ into SPLP, for the separation ofCPL-SPLP from free CPL. Profiles for lipid (), CPL (□), and DNA (⋄)with respect to the total amount applied to a Sepharose CL-4B column areshown. Panel B shows the column profile for Fraction #9 from Panel A.

[0085]FIG. 20 illustrates a time course for the insertion of CPL₄ (15nmol) into SPLP (200 mmol).

[0086]FIG. 21 illustrates a time course for the uptake of 20 μM of SPLPpossessing 0% (▪), 3% (⋄), or 4% () CPL₄ in BHK cells.

[0087]FIG. 22 illustrates transfection of BHK cells by SPLP (2.5 μg/mLpLuc) following insertion of various mol % of the CPL₄ compared to SPLPalone (0% CPL). Transfections were carried out by incubating the sampleson top of the cells for 4 or 9 hours and replacing with complete mediafor a complete 24 hours incubation (see also FIG. 33).

[0088]FIG. 23 tabulates CPL insertion results.

[0089]FIG. 24 also tabulates CPL insertion results.

[0090]FIG. 25 illustrates the post-insertion method for preparation ofCPL-containing liposomes. The preformed liposomes were made of DSPC/Chol(55:45, mol:mol). The CPL was incubated with the preformed liposomes at60° C. for 2 hour. Panel A illustrates separation of free CPL andCPL-LUVs by gel filtration after post-insertion. Panel B illustrateselution of fraction 10 (Panel A) on a Sepharose CL-4B column.

[0091]FIG. 26 illustrates cellular uptake of the stealth liposomescontaining DSPE-CPLs in BHK cells in DMEM (10% FBS). Control LUVs(DSPC/Chol/PEG-PE, 56:40:4) and CPL-LUVs (DSPC/Chol/PEG-PE/CPL,55.5:40:2:2) were prepared by extrusion as described herein.

[0092]FIG. 27 illustrates cellular uptake of stealth liposomescontaining DSPE-CPLs in BHK cells in PBS-CMG. Control LUVs(DSPC/Chol/PEG-PE, 56:40:4) and CPL-LUVs (DSPC/Chol/PEG-PE/CPL,55.5:40:2:2) were prepared by extrusion as described herein.

[0093]FIG. 28 Panel A: Chemical structures of various CPLs; Panel B:Chemical structures of various CPLs. Note that CPL₄ (Panel A) isidentical to CPL₄b (Panel B); and Panel C: Chemical structures ofvarious CPLs.

[0094]FIG. 29 illustrates a synthetic embodiment to generate compoundsof the present invention.

[0095]FIG. 30 illustrates a structure of dansylated CPL₄. CPL₄ possessesfour positive charges at the end of a PEG₃₄₀₀ molecule which is attachedto a DSPE molecule. The CPL₄ is dansylated by incorporation of adansylated lysine.

[0096]FIG. 31 illustrates an effect of cation concentration on thedeaggregation of SPLP-CPL₄. The mean diameter and standard deviation ofthe particles in the presence of increasing [Cation], Ca²⁺ () and Mg²⁺(▪), from 0 mM to 70 mM, was measured using quasi-elastic lightscattering (QELS). To ˜180 nmol of SPLP-CPL₄ in 400 mL in a Nicomp tubewas added small quantities of either CaCl₂ or MgCl₂ (500 mM stocksolutions). Measurement of the mean diameter±standard deviation of theparticles in the presence of differing amounts of the cation were madeusing a Nicomp Model 270 Submicron Particle Sizer. The diameters of theparticles do not dramatically change, however, the Gaussiandistributions do get broader. Thus, the standard deviations were used asa measure of deaggregation with smaller deviations indicating lessaggregation.

[0097]FIG. 32 illustrates uptake of SPLP containing various percentagesof CPL₄. Panel A. Time course for the uptake of 20 μM SPLP possessing 0mol % (), 2 mol % (▪), 3 mol % (▴), or 4 mol % (♦) CPL₄ and DOPE:DODACcomplexes (▾) by BHK cells. The insertion of the CPL₄ into SPLP and thepreparation of complexes was performed as described herein The mol % ofCPL₄ in the SPLP-CPL₄ was also determined, as described herein. BHKcells were plated in 12-well plates at 1×10⁵ cells/well. To 200 μL ofsample (containing SPLP-CPL₄ or complex+CaCl₂) was added 800 μL ofDMEM+10% FBS. The resulting CaCl₂ concentration was diluted to 20% ofthe original. Following incubation periods of 2, 4, 6 and 8 hours, thecells were lysed with 600 mL of lysis buffer and the rhodaminefluorescence and BCA assays were measured for the lysate, as describedherein (see FIG. 21).

[0098]FIG. 33 illustrates tansfection of BHK cells by SPLP (5.0 μg/mLpLuc) following insertion of various mole percentages of CPL₄ (2, 3, and4 mol %). The CPL₄ was inserted into SPLPs using the procedure describedherein. As a comparison, SPLP (0 mol % CPL) and DOPE:DODAC (1:1) complextransfections were also performed. BHK cells were plated at 1×10⁴ in96-well plates. Transfections were carried out by incubating the samples[20 μL (SPLP-CPL₄+CaCl₂)+80 μL of complete media] on the cells for 4hours followed by a 24 hour complete incubation. The CaCl₂ concentrationagain is diluted to 20% of the original concentration. Following the 24hour incubation, the cells were lysed with lysing buffer and theluciferase and BCA assays were performed (see FIG. 22).

[0099]FIG. 34 illustrates the effect of [Cation], Ca²⁺ () and Mg²⁺ (▪),on the transfection of SPLP-CLP₄ (5.0 μg/mL pLuc) on BHK cells.SPLP-CPL₄+CaCl₂ or MgCl₂ was mixed with DMEM+10% FBS and the mixtureswere applied to 1×10⁴ BHK cells plated in a 96-well plate. Following acomplete 48 hour incubation, the transfection media was removed and thecells were lysed with lysing buffer and the luciferase activity andprotein content were measured as described earlier.

[0100]FIG. 35 illustrates the effect of [Cation], Ca²⁺ () and Mg²⁺ (▪),on the lipid binding and uptake of 80 μM SPLP-CPL₄ on BHK cells. Thesamples possessing varying concentrations of the cation (0-14 mM finalconcentration) were incubated on 1×10⁵ BHK cells for 4 hours at whichtime the cells were lysed and the rhodamine fluorescence and proteincontent were measured.

[0101]FIG. 36 illustrates transfection of SPLP-CPL₄, SPLP and complexes(each containing 5.0 μg/mL pCMVLuc) at longer time points. Transfectionof SPLP-CPL₄ (4 mol % CPL₄)+40 mM_(initial) CaCl₂ (), SPLP (▾),DOPE:DODAC complexes (▪), and Lipofectin complexes (♦) was performed on1×10⁴ BHK cells. The transfection media was incubated on the cells for4, 8 or 24 hours, after which the transfection media was replaced bycomplete media for the 4 and 8 hour timepoints. Then at a totalincubation time of 24 hours (20, 16, and 0 hours, respectively, afterremoval of the transfection media), the cells were lysed and theluciferase activity and protein content were measured.

[0102]FIG. 37 illustrates transfection potency and toxicity of SPLP-CPL₄compared to Lipofectin complexes. A. Transfection activity forSPLP-CPL₄+CaCl₂ (), SPLP (▪), and Lipofectin (♦) on 1×10⁴ BHK cellsincubated for 24 and 48 hours followed by immediate cell lysis, andmeasurement of luciferase activity and protein content. B. Measurementof the cellular survival following 24 and 48 hour incubations of theSPLP-CPL₄+CaCl₂ (), Lipofectin (♦), and DOPE/DODAC (1:1) complexes on1×10⁴ BHK cells. Following incubation, the cells were lysed and theprotein content from the BCA assay was used as a measure of proteinsurvival.

[0103]FIG. 38 illustrates the transfection of BHK cells using both longand short chained CPLs. The presence of the short chained PEG in the CPLresults in a decrease by a factor of about 4 compared to thetransfection by the long chained CPL.

[0104]FIG. 39 illustrates the transfection of Neuro-2a cells. SPLP+4 mol% CPL4-1k produces 4 orders of magnitude of gene expression more thanSPLP alone in Neuro-2a cells.

[0105]FIG. 40 illustrates in vivo pharmacokinetics of SPLP containing ashort chain CPL₄.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS

[0106] A. Compounds and Synthesis

[0107] In certain aspects, the present invention providescationic-polymer-lipid conjugates (CPLs), such as distalcationic-poly(ethylene glycol)-lipid conjugates that can be incorporatedinto conventional and stealth liposomes for enhancing, inter alia,cellular uptake. The CPLs of the present invention have the followingarchitectural features: (1) a lipid anchor, such as a hydrophobic lipid,for incorporating the CPLs into the lipid bilayer; (2) a hydrophilicspacer, such as a polyethylene glycol, for linking the lipid anchor to acationic head group; and (3) a polycationic moiety, such as a naturallyoccurring amino acid, to produce a protonizable cationic head group. Assuch, the present invention provides a compound of Formula I:

A W-Y  I

[0108] wherein A, W and Y are as previously defined.

[0109] With reference to Formula I, “A” is a lipid moiety such as anamphipathic lipid, a neutral lipid or a hydrophobic lipid that acts as alipid anchor. Suitable lipid examples include vesicle-forming lipids orvesicle adopting lipids and include, but are not limited to,diacylglycerolyls, dialkylglycerolyls, N-N-dialkylaminos,1,2-diacyloxy-3-aminopropanes and 1,2-dialkyl-3-aminopropanes.

[0110] “W” is a polymer or an oligomer, such as a hydrophilic polymer oroligomer. Preferably, the hydrophilic polymer is a biocompatable polymerthat is non-immunogenic or possesses low inherent immunogenicity.Alternatively, the hydrophilic polymer can be weakly antigenic if usedwith appropriate adjuvants. Suitable non-immunogenic polymers include,but are not limited to, PEG, polyamides, polylactic acid, polyglycolicacid, polylactic acid/polyglycolic acid copolymers and combinationsthereof. In a preferred embodiment, the polymer has a molecular weightof about 250 to about 7000 daltons.

[0111] “Y” is a polycationic moiety. The term polycationic moiety refersto a compound, derivative, or functional group having a positive charge,preferably at least 2 positive charges at a selected pH, preferablyphysiological pH. Suitable polycationic moieties include basic aminoacids and their derivatives such as arginine, asparagine, glutamine,lysine and histidine; spermine; spermidine; cationic dendrimers;polyamines; polyamine sugars; and amino polysaccharides. Thepolycationic moieties can be linear, such as linear tetralysine,branched or dendrimeric in structure. Polycationic moieties have betweenabout 2 to about 15 positive charges, preferably between about 2 toabout 12 positive charges, and more preferably between about 2 to about8 positive charges at selected pH values. The selection of whichpolycationic moiety to employ may be determined by the type of liposomeapplication which is desired.

[0112] The charges on the polycationic moieties can be eitherdistributed around the entire liposome moiety, or alternatively, theycan be a discrete concentration of charge density in one particular areaof the liposome moiety e.g., a charge spike. If the charge density isdistributed on the liposome, the charge density can be equallydistributed or unequally distributed. All variations of chargedistribution of the polycationic moiety are encompassed by the presentinvention.

[0113] The lipid “A”, and the non-immunogenic polymer “W”, can beattached by various methods and preferably, by covalent attachment.Methods known to those of skill in the art can be used for the covalentattachment of “A” and “W”. Suitable linkages include, but are notlimited to, amide, amine, carboxyl, carbonate, carbamate, ester andhydrazone linkages. It will be apparent to those skilled in the art that“A” and “W” must have complementary functional groups to effectuate thelinkage. The reaction of these two groups, one on the lipid and theother on the polymer, will provide the desired linkage. For example,when the lipid is a diacylglycerol and the terminal hydroxyl isactivated, for instance with NHS and DCC, to form an active ester, andis then reacted with a polymer which contains an amino group, such aswith a polyamide (see, U.S. patent application Ser. No. 09/218,988,filed Dec. 22, 1998), an amide bond will form between the two groups.

[0114] In certain embodiments, “W” is bound, preferably covalentlybound, to “Y”. As with “A” and “W”, a covalent attachment of “W” to “Y”can be generated by complementary reactivity of functional groups, oneon the polymer and the other on the polycationic moiety. For example, anamine functional group on “W” can be reacted with an activated carboxylgroup, such as an acyl chloride or NHS ester, to form an amide. Bysuitable choice of reactive groups, the desired coupling can beobtained. Other activated carboxyl groups include, but are not limitedto, a carboxylic acid, a carboxylate ester, a carboxylic acid halide andother activated forms of carboxylic acids, such as a reactive anhydride.Reactive acid halides include for example, acid chlorides, acidbromides, and acid fluorides.

[0115] In certain instances, the polycationic moiety can have a ligandattached, such as a targeting ligand. Preferably, after the ligand isattached, the cationic moiety maintains a positive charge. In certaininstances, the ligand that is attached has a positive charge. Suitableligands include, but are not limited to, a compound or device with areactive functional group and includes lipids, amphipathic lipids,carrier compounds, bioaffinity compounds, biomaterials, biopolymers,biomedical devices, analytically detectable compounds, therapeuticallyactive compounds, enzymes, peptides, proteins, antibodies, immunestimulators, radiolabels, fluorogens, biotin, drugs, haptens, DNA, RNA,polysaccharides, liposomes, virosomes, micelles, immunoglobulins,functional groups, other targeting moieties, or toxins.

[0116] In certain preferred embodiments, other moieties are incorporatedinto the compounds of Formula I to form the compounds of Formula II:

[0117] In Formula II, “A” is a lipid moiety such as, an amphipathiclipid, a neutral lipid or a hydrophobic lipid moiety. Suitable lipidexamples include, but are not limited to, diacylglycerolyl,dialkylglycerolyl, N-N-dialkylamino, 1,2-diacyloxy-3-aminopropane and1,2-dialkyl-3-aminopropane.

[0118] In Formula II, “X” is a single bond or a functional group thatcovalently attaches the lipid to at least one ethylene oxide unit.Suitable functional groups include, but are not limited to,phosphatidylethanolamino, phosphatidylethanolamido, phosphoro, phospho,phosphoethanolamino, phosphoethanolamido, carbonyl, carbamate, carboxyl,carbonate, amido, thioamido, oxygen, NR wherein R is a hydrogen or alkylgroup and sulfur. In certain instances, the lipid “A” is directlyattached to the ethylene oxide unit by a single bond. The number ofethylene oxide units can range from about 1 to about 160 and preferablyfrom about 6 to about 50.

[0119] In Formula II, “Z” is a single bound or a functional group thatcovalently attaches the ethylene oxide unit to the polycationic moiety.Suitable functional groups include, but are not limited to, phospho,phosphoethanolamino, phosphoethanolamido, carbonyl, carbamate, carboxyl,amido, thioamido, NR wherein R is a member selected from the groupconsisting of hydrogen atom or alkyl group. In certain embodiments, theterminal ethylene oxide unit is directly attached to the polycationicmoiety.

[0120] In Formula II, “Y” is a polycationic moiety as described above inconnection with Formula I. In Formula II, the index “n” is an integerranging in value from about 6 to about 160.

[0121] In an illustrative embodiment, compounds of Formula II can besynthesized using a generalized procedure as outlined in FIG. 2. FIG. 2illustrates one particular embodiment of the present invention and thus,is merely an example that should not limit the scope of the claimsherein. Clearly, one of ordinary skill in the art will recognize manyother variations, alternatives, and modifications that can be made tothe reaction scheme illustrated in FIG. 2. With reference to FIG. 2, asolution of a lipid, such as DSPE, and a base, such as triethylamine ina chloroform solution is added to (t-Boc-NH-PEG₃₄₀₀-CO₂NHS), and thesolution is stirred at ambient temperature. The solution is thenconcentrated under a nitrogen stream to dryness. The residue is thenpurified by repeated precipitation of the chloroform mixture solutionwith diethyl ether until disappearance of the lipid usingchromatography. The purified CPL conjugate is dissolved in a solvent,followed by addition of TFA, and the solution is stirred at roomtemperature. The solution can again be concentrated under a nitrogenstream. The residue is then purified by repeated precipitation of themixture with diethyl ether to offer a lipid-PEG-NH₂, such as aDSPE-PEG-NH₂ or, alternatively, DSPE-CPL-1 with one protonizablecationic head group. The ratio of the phosphoryl-lipid anchor and thedistal primary amine can then be measured by phosphate and flourescamineassays as described herein.

[0122] In this illustrative embodiment, the number of protonizable aminogroups can be increased to create a polycationic moiety. Byincrementally adding stoichiometric amounts of, for example, aNα,Nε-di-t-Boc-L-Lysine N-hydroxysuccinide ester, the polycationicmoiety can be increase from about 2 to about 16 positive charges. Asdescribe previously, the positive charges can be incorporated using anynumber of suitable polycationic moieties such as lysine, arginine,asparagine, glutamine, histidine, polyamines and derivatives orcombinations thereof. Using the synthesis methods of the presentinvention, the number of cationic groups, such as amino groups, can bereadily controlled during the CPL synthesis.

[0123] B. Lipid-Based Drug Formulations

[0124] In certain aspects, the present invention provides a lipid-baseddrug formulation comprising:

[0125] (a) a compound having the general structure of Formula I:

A-W-Y  I

[0126] wherein A, W and Y are as previously defined; (b) a bioactiveagent; and optionally, (c) a second lipid. In preferred embodiments, thelipid-based drug formulation of the present invention comprises thesecond lipid, such as a PEG-lipid derivative.

[0127] In certain preferred embodiments, the lipid-based drugformulations of the present invention comprise

[0128] (a) a compound of Formula II:

[0129] wherein A, X, Z, Y and n have been previously defined; (b) abioactive agent; and optionally, (c) a second lipid. In preferredembodiments, the lipid-based drug formulation of the present inventioncomprises the second lipid, such as a PEG-lipid derivative.

[0130] After the CPLs have been prepared, they can be utilized in avariety of ways including, for example, in lipid-based drugformulations. In this aspect, the lipid-based formulations can be in theform of a liposome, a micelle, a virosome, a lipid-nucleic acidparticle, a nucleic acid aggregate and other forms which can incorporateor entrap one or more bioactive agents. In certain aspects, thelipid-based drug formulations of the present invention comprise a secondlipid.

[0131] The compounds of Formulae I and II can be used in lipid-basedformulations such as those described in for example, the followingcopending U.S. patent application Ser. Nos. 08/454,641, 08/485,458,08/660,025, 08/484,282, 60/055,094, 08/856,374, 60/053,813 and60/063,473, entitled “Methods for Encapsulating Nucleic Acids in LipidBilayers,” filed on Oct. 10, 1997 and bearing Attorney Docket No.016303-004800, U.S. Pat. No. 5,703,055, U.S. patent application Ser. No.09/218,988, filed Dec. 22, 1998, the teachings all of which areincorporated herein by reference in their entirety for all purposes.This specification sets out a variety of liposome types and a variety ofmethods for incorporating CPLs into liposomes, all of which are examplesof the broad methods and compositions claimed herein.

[0132] The lipid components and CPLs used in forming the variouslipid-based drug formulations will depend, in part, on the type ofdelivery system employed. For instance, if a liposome is employed, thelipids used in the CPL will generally be selected from a variety ofvesicle-forming or vesicle-adopting lipids, typically includingphospholipids and sterols, such as phosphatidylenthanolamine (PE),phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol(PG), phosphatidic acid (PA), which have been suitably functionalized,and the like. In contrast, if a micelle is employed, the lipids used inthe CPL will generally be selected from sterylamines, alkylamines,C₈-C₂₂ alkanoic acids, lysophospholipids, detergents and the like. Itwill be readily apparent to those of skill in the art that the acylchains can be varied in length and can be saturated or possess varyingdegrees of unsaturation. The more saturated the acyl chains the morerigid the membrane. Higher degrees of unsaturation impart more fluidityinto the vesicle's membrane. Similarly, the other lipid components(e.g., lipids, cationic lipids, neutral lipids, non-cationic lipids,etc.) making up the drug delivery systems of the present invention willvary depending on the drug delivery system employed. Suitable lipids forthe various drug delivery systems will be readily apparent to those ofskill in the art.

[0133] When the lipid-based drug formulations are used to delivertherapeutic genes or oligonucleotides intended to induce or to blockproduction of some protein within the cell, cationic lipids can beincluded in the formulation, e.g., liposome, micelle, lipid-nucleic acidparticle, etc. Nucleic acid is negatively charged and can be combinedwith a positively charged entity to form a lipid complex suitable forformulation and cellular delivery.

[0134] As used in this specification, “cationic lipid” generally refersto a lipid with a cationic head group situated at or near the liposomemembrane (when incorporated in a liposome). CPLs are distinguished fromcationic lipids by the polymer “W” which in certain instances, has theeffect of placing the cationic charge at a significant distance from themembrane.

[0135] Examples of suitable cationic lipids include, but are not limitedto, the following: DC-Chol, (see, Gao, et al., Biochem. Biophys. Res.Comm., 179:280-285 (1991); DDAB; DMRIE; DODAC (see, U.S. patentapplication Ser. No. 08/316,399, filed Sep. 30, 1994, which isincorporated herein by reference); DOGS; DOSPA; DOTAP; and DOTMA. In apresently preferred embodiment, N,N-dioleoyl-N,N-dimethylammoniumchloride is used in combination with a phosphatidylethanolamine.

[0136] In addition, other cationic lipids useful in producinglipid-based carriers for gene and oligonucleotide delivery areLIPOFECTIN (U.S. Pat. Nos. 4,897,355; 4,946,787; and 5,208,036 issued toEppstein, et al.) and LIPOFECTACE (U.S. Pat. No. 5,279,883 issued toRose). Both agents, as well as other transfecting cationic lipids, areavailable from Life Technologies, Inc. in Gaithersburg, Md.

[0137] In one preferred embodiment, the CPL-liposomes of the presentinvention are optimized for systemic delivery applications. In certainapplications, the polymer length in the CPL is shorter than the normalneutral PEG chains (M.W. 2000-5000 Daltons) used for stealth liposomes.In this instance, the shorter polymer in the CPL is about 250 to about3000 Daltons and more preferably, about 1000 to about 2000 Daltons. Inthis embodiment, the second lipid is for example, a PEG₃₄₀₀-lipid andthe compound of Formula I is, for example, A-PEG₁₀₀₀-Y. (see, FIG. 17C).

[0138] Without being bound by any particular theory, when the shorterpolymer is used, it is believed that the distal charge(s) of the CPL ishidden within the normal PEG exclusion barrier, thus allowing retentionof long circulation lifetimes while at the same time, extending thepositive charges away from the liposomal surface. This embodimentenhances interactions between liposomes and a target cell. The use ofdifferent sized polymers, such as PEG chains, in the CPLs and theneutral PEG-lipids used to modulate vesicle circulation and cellularuptake, allows for a new generation of stealth liposomes as drugcarriers. It is believed that the optimized polymer length can vary withthe specific conditions such as in vitro or in vivo applications, localor systemic administration, and different lipid formulations.

[0139] In another embodiment, the polymer length in the CPL has a largerMW than the normal neutral PEG chains used for stealth liposomes. Inthis instance, the second lipid is for example, a PEG₁₀₀₀-lipid and thecompound of Formula I has a formula of, for example, A-PEG₃₄₀₀-Y. (see,FIG. 17B).

[0140] In certain formulations and applications, the type of CPL i.e.the length of the polymer chain, and the amount of cationic charge permolecule, and the amount of such CPL in a formulation e.g., SPLP, can beoptimized to obtain the best balancing of clearance properties. Incertain instances, long chain CPLs and higher levels of such CPLs are tobe preferred to increase transfection. In other instances, short chainCPLs incorporated in the formulations are optimized for longercirculation lifetimes in animals.

[0141] In one embodiment of the present invention, a fusogenic liposomeor virosome is provided. It will be readily apparent to those of skillin the art that the CPLs of the present invention can advantageously beincorporated into various types of fusogenic liposomes and virosomes.Such fusogenic liposomes and virosomes can be designed to becomefusogenic at the disease or target site. Those of skill in the art willreadily appreciate that a number of variables can be used to controlwhen the liposome or virosome becomes fusogenic. Such variables include,for example, the composition of the liposome or virosome, pH,temperature, enzymes, cofactors, ions, etc.

[0142] In one embodiment, the fusogenic liposome comprises: a lipidcapable of adopting a non-lamellar phase, yet capable of assuming abilayer structure in the presence of a bilayer-stabilizing component(such as a PEG-lipid derivative); and a bilayer-stabilizing componentreversibly associated with the lipid to stabilize the lipid in a bilayerstructure. Such fusogenic liposomes are advantageous because the rate atwhich they become fusogenic can be not only predetermined, but varied asrequired over a time scale of a few minutes to several tens of hours. Ithas been found, for example, that by controlling the composition andconcentration of the bilayer-stabilizing component, one can control therate at which the BSC exchanges out of the liposome in vivo and, inturn, the rate at which the liposome becomes fusogenic (see, U.S. Pat.No. 5,885,613). For instance, it has been found that by controlling thelength of the lipid acyl chain(s), one can control the rate at which theBSC exchanges out of the liposome in vivo and, in turn, the rate atwhich the liposome becomes fusogenic. In particular, it has beendiscovered that shorter acyl chains (e.g., C-8) exchange out of theliposome more rapidly than longer acyl chains (e.g., C-20).Alternatively, by controlling the composition and concentration of theBSC, one can control the rate at which the BSC is degraded, i.e., brokendown, by endogenous systems, e.g., endogenous enzymes in the serum, and,in turn, the rate at which the liposome becomes fusogenic.

[0143] The polymorphic behavior of lipids in organized assemblies can beexplained qualitatively in terms of the dynamic molecular shape concept(see, Cullis, et al., in “Membrane Fusion” (Wilschut, J. and D. Hoekstra(eds.), Marcel Dekker, Inc., New York, (1991)). When the effectivecross-sectional areas of the polar head group and the hydrophobic regionburied within the membrane are similar then the lipids have acylindrical shape and tend to adopt a bilayer conformation. Cone-shapedlipids which have polar head groups that are small relative to thehydrophobic component, such as unsaturated phosphatidylethanolamines,prefer non-bilayer phases such as inverted micelles or inverse hexagonalphase (H ). Lipids with head groups that are large relative to theirhydrophobic domain, such as lysophospholipids, have an inverted coneshape and tend to form micelles in aqueous solution. The phasepreference of a mixed lipid system depends, therefore, on thecontributions of all the components to the net dynamic molecular shape.As such, a combination of cone-shaped and inverted cone-shaped lipidscan adopt a bilayer conformation under conditions where either lipid inisolation cannot (see, Madden and Cullis, Biochim. Biophys. Acta,684:149-153 (1982)).

[0144] A more formalized model is based on the intrinsic curvaturehypothesis (see, e.g., Kirk, et al., Biochemistry, 23:1093-1102 (1984)).This model explains phospholipid polymorphism in terms of two opposingforces. The natural tendency of a lipid monolayer to curl and adopt itsintrinsic or equilibrium radius of curvature (R_(O)) which results in anelastically relaxed monolayer is opposed by the hydrocarbon packingconstraints that result. Factors that decrease the intrinsic radius ofcurvature, such as increased volume occupied by the hydrocarbon chainswhen double bonds are introduced, tend to promote H phase formation.Conversely, an increase in the size of the headgroup increases R_(O) andpromotes bilayer formation or stabilization. Introduction of apolarlipids that can fill the voids between inverted lipid cylinders alsopromotes H phase formation (see, Gruner, et al., Proc. Natl. Acad. Sci.USA, 82:3665-3669 (1989); Sjoland, et al., Biochemistry, 28:1323-1329(1989)).

[0145] As such, in one embodiment, the lipids which can be used to formthe fusogenic liposomes of the present invention are those which adopt anon-lamellar phase under physiological conditions or under specificphysiological conditions, e.g., in the presence of calcium ions, butwhich are capable of assuming a bilayer structure in the presence of aBSC. Such lipids include, but are not limited to,phosphatidylenthanolamines, ceramides, glycolipids, or mixtures thereof.Other lipids known to those of skill in the art to adopt a non-lamellarphase under physiological conditions can also be used. Moreover, it willbe readily apparent to those of skill in the art that other lipids canbe induced to adopt a non-lamellar phase by various non-physiologicalchanges including, for example, changes in pH or ion concentration(e.g., in the presence of calcium ions) and, thus, they can also be usedto form the fusogenic liposomes of the present invention. In a presentlypreferred embodiment, the fusogenic liposome is prepared from aphosphatidylethanolamine. The phosphatidylethanolamine can be saturatedor unsaturated. In a presently preferred embodiment, thephosphatidylyethanolamine is unsaturated. In an equally preferredembodiment, the fusogenic liposome is prepared from a mixture of aphosphatidylethanolamine (saturated or unsaturated) and aphosphatidylserine. In another equally preferred embodiment, thefusogenic liposome is prepared from a mixture of aphosphatidylethanolamine (saturated or unsaturated) and a cationiclipid.

[0146] In one embodiment, the lipid-based drug formulations of thepresent invention comprise a bilayer stabilizing component (BSC).Suitable BSCs include, but are not limited to, polyamide oligomers,peptides, proteins, detergents, lipid-derivatives, PEG-lipids such asPEG coupled to phosphatidylethanolamine, and PEG conjugated to ceramides(see, U.S. Pat. No. 5,885,613, which is incorporated herein byreference). Preferably, the bilayer stabilizing component is aPEG-lipid, or an ATTA-lipid. As discussed herein, in certain preferredinstances, the PEG or the ATTA of the BSC has a greater molecular weightcompared to the polymer “W” of the CPL. In other instances, the BSC hasa smaller molecular weight compared to the “W” of the polymer. Thepresent invention encompasses all such variations.

[0147] In accordance with the present invention, lipids adopting anon-lamellar phase under physiological conditions can be stabilized in abilayer structure by BSCs which are either bilayer forming themselves,or which are of a complementary dynamic shape. The non-bilayer forminglipid is stabilized in the bilayer structure only when it is associatedwith, i.e., in the presence of, the BSC. In selecting an appropriateBSC, it is preferable that the BSC be capable of transferring out of theliposome, or of being chemically modified by endogenous systems suchthat, with time, it loses its ability to stabilize the lipid in abilayer structure. Only when liposomal stability is lost or decreasedcan fusion of the liposome with the plasma membrane of the target celloccur. The BSC-lipid, therefore, is “reversibly associated” with thelipid and only when it is associated with the lipid is the lipidconstrained to adopt the bilayer structure under conditions where itwould otherwise adopt a non-lamellar phase. As such, the BSC-lipids ofthe present invention are capable of stabilizing the lipid in a bilayerstructure, yet they are capable of exchanging out of the liposome, or ofbeing chemically modified by endogenous systems so that, with time, theylose their ability to stabilize the lipid in a bilayer structure,thereby allowing the liposome to become fusogenic.

[0148] Typically, the CPL is present in the lipid-based formulation ofthe present invention at a concentration ranging from about 0.05 molepercent to about 50 mole percent. In a presently preferred embodiment,the CPL is present at a concentration ranging from 0.05 mole percent toabout 25 mole percent. In an even more preferred embodiment, the CPL ispresent at a concentration ranging from 0.05 mole percent to about 15mole percent. One of ordinary skill in the art will appreciate that theconcentration of the CPL can be varied depending on the CPL employed andthe rate at which the liposome is to become fusogenic.

[0149] In one embodiment of the present invention, the liposomes containcholesterol. It has been determined that when cholesterol-free liposomesare used in vivo, they have a tendency to absorb cholesterol from theplasma lipoproteins and cell membranes. Cholesterol, if included, isgenerally present at a concentration ranging from 0.2 mole percent toabout 50 mole percent and, more preferably, at a concentration rangingfrom about 35 mole percent to about 45 mole percent.

[0150] C. Preparation of CPL-Liposomes

[0151] A variety of general methods for making CPL-containing liposomes(or “CPL-liposomes”) are discussed herein.

[0152] Two general techniques include “post-insertion,” that is,insertion of a CPL into for example, a pre-formed liposome vesicle, and“standard” techniques, wherein the CPL is included in the lipid mixtureduring for example, the liposome formation steps. The post-insertiontechnique results in liposomes having CPLs mainly in the external faceof the liposome bilayer membrane, whereas standard techniques provideliposomes having CPLs on both internal and external faces.

[0153] In particular, “post-insertion” involves forming vesicles (by anymethod), and incubating the pre-formed vesicles in the presence of CPLunder appropriate conditions (usually 2-3 hours at 60° C.). Between60-80% of the CPL can be inserted into the external leaflet of therecipient vesicle, giving final concentrations up to 7 mol % (relativeto total lipid). The method is especially useful for vesicles made fromphospholipids (which can contain cholesterol) and also for vesiclescontaining PEG-lipids (such as PEG-Ceramide).

[0154] In an example of a “standard” technique, the CPL-LUVs of thepresent invention can be formed by extrusion. In this embodiment, all ofthe lipids including CPL, are co-dissolved in chloroform, which is thenremoved under nitrogen followed by high vacuum. The lipid mixture ishydrated in an appropriate buffer, and extruded through twopolycarbonate filters with a pore size of 100 nm. The resulting vesiclescontain CPL on both internal and external faces. In yet another standardtechnique, the formation of CPL-LUVs can be accomplished using adetergent dialysis or ethanol dialysis method, for example, as discussedin U.S. Pat. Nos. 5,976,567 and 5,981,501, both of which areincorporated herein by reference. The extrusion method and the detergentdialysis method are explained in detail in the Example section.

[0155] D. Liposome Preparation and Sizing

[0156] A variety of methods are available for preparing and sizingliposomes as described in, e.g., Szoka, et al., Ann. Rev. Biophys.Bioeng., 9:467 (1980), U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871,4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, 4,946,787, PCTPublication No. WO 91/17424, Deamer and Bangham, Biochim. Biophys. Acta,443:629-634 (1976); Fraley, et al., Proc. Natl. Acad. Sci. USA,76:3348-3352 (1979); Hope, et al., Biochim. Biophys. Acta, 812:55-65(1985); Mayer, et al., Biochim. Biophys. Acta, 858:161-168 (1986);Williams, et al., Proc. Natl. Acad. Sci., 85:242-246 (1988), the textLiposomes, Marc J. Ostro, ed., Marcel Dekker, Inc., New York, 1983,Chapter 1, and Hope, et al., Chem. Phys. Lip., 40:89 (1986), all ofwhich are incorporated herein by reference. Suitable methods include,but are not limited to, sonication, extrusion, highpressure/homogenization, microfluidization, detergent dialysis,calcium-induced fusion of small liposome vesicles, and ether-infusionmethods, all of which are well known in the art. One method producesmultilamellar vesicles of heterogeneous sizes. In this method, thevesicle-forming lipids are dissolved in a suitable organic solvent orsolvent system and dried under vacuum or an inert gas to form a thinlipid film. If desired, the film may be redissolved in a suitablesolvent, such as tertiary butanol, and then lyophilized to form a morehomogeneous lipid mixture which is in a more easily hydrated powder-likeform. This film is covered with an aqueous buffered solution and allowedto hydrate, typically over a 15-60 minute period with agitation. Thesize distribution of the resulting multilamellar vesicles can be shiftedtoward smaller sizes by hydrating the lipids under more vigorousagitation conditions or by adding solubilizing detergents, such asdeoxycholate.

[0157] Extrusion of liposome through a small-pore polycarbonate membraneor an asymmetric ceramic membrane is an effective method for reducingliposome sizes to a relatively well-defined size distribution.Typically, the suspension is cycled through the membrane one or moretimes until the desired liposome size distribution is achieved. Theliposomes may be extruded through successively smaller-pore membranes,to achieve gradual reduction in liposome size. For use in the presentinvention, liposomes having a size ranging from about 0.05 microns toabout 0.40 microns are preferred.

[0158] E. Use of Liposomes as Drug Delivery Vehicles

[0159] The lipid-based drug formulations and compositions of the presentinvention (e.g., liposomes, micelles, lipid-nucleic acid particles,virosomes, etc.) are useful for the systemic or local delivery ofbioactive agents such as therapeutic agents, prophylactic agents anddiagnostic agents. Such delivery systems are described in greater detailin, for example, the following copending U.S. patent application Ser.Nos. 08/454,641, 08/485,458, 08/660,025, 08/484,282, 60/055,094,08/856,374, 60/053,813 and 60/063,473, the teachings of all of which areincorporated herein by reference.

[0160] The following discussion refers generally to liposomes; however,it will be readily apparent to those of skill in the art that this samediscussion is fully applicable to the other drug delivery systems of thepresent invention (e.g., micelles, virosomes, lipid-nucleic acidparticles, etc.).

[0161] For the delivery of therapeutic agents, the compositions can beloaded with a therapeutic agent and administered to the subjectrequiring treatment. The therapeutic agents which are administered usingthe present invention can be any of a variety of drugs which areselected to be an appropriate treatment for the disease to be treated orprevented. Often the drug will be an antineoplastic agent, such asvincristine, doxorubicin, mitoxantrone, camptothecin, cisplatin,bleomycin, cyclophosphamide, methotrexate, streptozotocin, and the like.Especially preferred antitumor agents include, for example, actinomycinD, vincristine, vinblastine, cystine arabinoside, anthracyclines,alkylative agents, platinum compounds, antimetabolites, and nucleosideanalogs, such as methotrexate and purine and pyrimidine analogs. It mayalso be desirable to deliver anti-infective agents to specific tissuesby the present methods. The compositions of the present invention canalso be used for the selective delivery of other drugs including, butnot limited to, local anesthetics, e.g., dibucaine and chlorpromazine;beta-adrenergic blockers, e.g., propranolol, timolol and labetolol;antihypertensive agents, e.g., clonidine and hydralazine;anti-depressants, e.g., imipramine, amitriptyline and doxepim;anti-conversants, e.g., phenyloin; antihistamines, e.g.,diphenhydramine, chlorphenirimine and promethazine;antibiotic/antibacterial agents, e.g., gentamycin, ciprofloxacin, andcefoxitin; antifungal agents, e.g., miconazole, terconazole, econazole,isoconazole, butaconazole, clotrimazole, itraconazole, nystatin,naftifine and amphotericin B; antiparasitic agents, hormones, hormoneantagonists, immunomodulators, neurotransmitter antagonists,antiglaucoma agents, vitamins, narcotics, and imaging agents.

[0162] As mentioned above, cationic lipids can be used in the deliveryof therapeutic genes or oligonucleotides intended to induce or to blockproduction of some protein within the cell. Nucleic acid is negativelycharged and may be combined with a positively charged entity to form alipid complex or a fully encapsulated stable plasmid-lipid particle.

[0163] Particularly useful antisense oligonucleotides are directed totargets such as c-myc, bcr-abl, c-myb, ICAM-1, C-erb B-2 and BCL-2.

[0164] The CPLs of the present invention are also useful in the deliveryof peptides, nucleic acids, plasmid DNA, minichromosomes and ribozymes.

[0165] Another clinical application of CPLs of this invention is as anadjuvant for immunization of both animals and humans. Protein antigens,such as diphtheria toxoid, cholera toxin, parasitic antigens, viralantigens, immunoglobulins, enzymes and histocompatibility antigens, canbe incorporated into or attached onto the liposomes containing the CPLsof the present invention for immunization purposes.

[0166] Liposomes containing the CPLs of the present invention are alsoparticularly useful as carriers for vaccines that will be targeted tothe appropriate lymphoid organs to stimulate an immune response.

[0167] Liposomes containing the CPLs of the present invention can alsobe used as a vector to deliver immunosuppressive or immunostimulatoryagents selectively to macrophages. In particular, glucocorticoids usefulto suppress macrophage activity and lymphokines that activatemacrophages can be delivered using the liposomes of the presentinvention.

[0168] Liposomes containing the CPLs of the present invention andcontaining targeting molecules can be used to stimulate or suppress acell. For example, liposomes incorporating a particular antigen can beemployed to stimulate the B cell population displaying surface antibodythat specifically binds that antigen. Liposomes incorporating growthfactors or lymphokines on the liposome surface can be directed tostimulate cells expressing the appropriate receptors for these factors.Using this approach, bone marrow cells can be stimulated to proliferateas part of the treatment of cancer patients.

[0169] Liposome-encapsulated antibodies can be used to treat drugoverdoses. The tendency of liposomes having encapsulated antibodies tobe delivered to the liver has a therapeutic advantage in clearingsubstances, such as toxic agents, from the blood circulation. It hasbeen demonstrated that whereas unencapsulated antibodies to digoxincaused intravascular retention of the drug, encapsulated antibodiescaused increased splenic and hepatic uptake and an increased excretionrate of digoxin.

[0170] Liposomes containing the CPLs of this invention also find utilityas carriers for introducing lipid or protein antigens into the plasmamembrane of cells that lack the antigens. For example,histocompatibility antigens or viral antigens can be introduced into thesurface of viral infected or tumor cells to promote recognition andkilling of these cells by the immune system.

[0171] In addition, liposomes containing the CPLs of the presentinvention can be used to deliver any product (e.g., therapeutic agents,diagnostic agents, labels or other compounds) including those currentlyformulated in PEG-derivatized liposomes.

[0172] In certain embodiments, it is desirable to target the liposomesof this invention using targeting moieties that are specific to a celltype or tissue. Targeting of liposomes using a variety of targetingmoieties, such as ligands, cell surface receptors, glycoproteins,vitamins (e.g., riboflavin) and monoclonal antibodies, has beenpreviously described (see, e.g., U.S. Pat. Nos. 4,957,773 and 4,603,044,the teachings of which are incorporated herein by reference). Thetargeting moieties can comprise the entire protein or fragments thereof.

[0173] In some cases, the diagnostic targeting of the liposome cansubsequently be used to treat the targeted cell or tissue. For example,when a toxin is coupled to a targeted liposome, the toxin can then beeffective in destroying the targeted cell, such as a neoplasmic cell.

[0174] In another aspect, the present invention provides a method forincreasing intracellular delivery of a lipid-based drug formulation,comprising: incorporating into the lipid-based drug formulation, acompound of Formulae I or II, thereby increasing the intracellulardelivery of the lipid based drug formulation compared to a formulationwithout a compound of Formulae I or II. The compounds of Formulae I orII increase intracellular delivery about 10 fold to about 1000 fold andpreferably, about 10 fold to about 100000 fold.

[0175] In another aspect, the present invention provides a method ofincreasing the blood-circulation time of a parenterally administeredlipid-based drug formulation, the method comprising: incorporating intothe lipid-based drug formulation about 0.1 to 20 mole percent of acompound of Formulae I or II.

[0176] In other aspects, the present invention provides a method fortransfection of a cell with a lipid-based drug formulation, comprising:contacting the cell with a lipid-based drug formulation having about 0.1to 20 mole percent of a compound of Formulae I or II. Moreover, a methodfor increasing the transfection of a cell with a lipid-based drugformulation, comprising: contacting the cell with a lipid-based drugformulation having about 0.1 to 20 mole percent of a compound ofFormulae I or II, whereby the transfection efficiency of the lipid-baseddrug formulation is increased compared to the transfection efficiency ofa lipid-based drug formulation without the compound of Formulae I or II.

[0177] G. Use of the Liposomes as Diagnostic Agents

[0178] The lipid-based drug formulations or compositions, e.g.,liposomes, prepared using the CPLs of this invention can be labeled withmarkers that will facilitate diagnostic imaging of various diseasestates including tumors, inflamed joints, lesions, etc. Typically, theselabels will be radioactive markers, although fluorescent labels can alsobe used. The use of gamma-emitting radioisotopes is particularlyadvantageous as they can easily be counted in a scintillation wellcounter, do not require tissue homogenization prior to counting and canbe imaged with gamma cameras.

[0179] Gamma- or positron-emitting radioisotopes are typically used,such as ⁹⁹Tc, ²⁴Na, ⁵¹Cr, ⁵⁹Fe, ⁶⁷Ga, ⁸⁶Rb, ¹¹¹In, ¹²⁵I, and ¹⁹⁵Pt asgamma-emitting; and such as ⁶⁸Ga, ⁸²Rb, ²²Na ⁷⁵Br, ¹²²I and ¹⁸F aspositron-emitting.

[0180] The liposomes can also be labelled with a paramagnetic isotopefor purposes of in vivo diagnosis, as through the use of magneticresonance imaging (MRI) or electron spin resonance (ESR). See, forexample, U.S. Pat. No. 4,728,575, the teachings of which areincorporated herein by reference.

[0181] H. Loading and Administering the Liposomes

[0182] The following discussion refers generally to liposomes; however,it will be readily apparent to those of skill in the art that this samediscussion is fully applicable to the other drug delivery systems of thepresent invention (e.g., micelles, virosomes, lipid-nucleic acidparticles, etc.). Methods of loading conventional drugs into liposomesinclude, for example, an encapsulation technique, loading into thebilayer and a transmembrane potential loading method.

[0183] In one encapsulation technique, the drug and liposome componentsare dissolved in an organic solvent in which all species are miscibleand concentrated to a dry film. A buffer is then added to the dried filmand liposomes are formed having the drug incorporated into the vesiclewalls. Alternatively, the drug can be placed into a buffer and added toa dried film of only lipid components. In this manner, the drug willbecome encapsulated in the aqueous interior of the liposome. The bufferwhich is used in the formation of the liposomes can be any biologicallycompatible buffer solution of, for example, isotonic saline, phosphatebuffered saline, or other low ionic strength buffers. Generally, thedrug will be present in an amount of from about 0.01 ng/mL to about 50mg/mL. The resulting liposomes with the drug incorporated in the aqueousinterior or in the membrane are then optionally sized as describedabove.

[0184] Transmembrane potential loading has been described in detail inU.S. Pat. No. 4,885,172, U.S. Pat. No. 5,059,421, and U.S. Pat. No.5,171,578, the contents of which are incorporated herein by reference.Briefly, the transmembrane potential loading method can be used withessentially any conventional drug which can exist in a charged statewhen dissolved in an appropriate aqueous medium. Preferably, the drugwill be relatively lipophilic so that it will partition into theliposome membranes. A transmembrane potential is created across thebilayers of the liposomes or protein-liposome complexes and the drug isloaded into the liposome by means of the transmembrane potential. Thetransmembrane potential is generated by creating a concentrationgradient for one or more charged species (e.g., Na⁺, K⁺ and/or H⁺)across the membranes. This concentration gradient is generated byproducing liposomes having different internal and external media and hasan associated proton gradient. Drug accumulation can than occur in amanner predicted by the Henderson-Hasselbach equation.

[0185] The liposome compositions of the present invention can byadministered to a subject according to standard techniques. Preferably,pharmaceutical compositions of the liposome compositions areadministered parenterally, i.e., intraperitoneally, intravenously,subcutaneously or intramuscularly. More preferably, the pharmaceuticalcompositions are administered intravenously by steady infusion. Suitableformulations for use in the present invention are found in Remington'sPharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa.,17th ed. (1985). The pharmaceutical compositions can be used, forexample, to diagnose a variety of conditions, or treat a diseased state.The diseases include, but are not limited to, inflammation associatedwith rheumatoid arthritis, post-ischemic leukocyte-mediated tissuedamage (reperfusion injury), acute leukocyte-mediated lung injury (e.g.,adult respiratory distress syndrome), septic shock, and acute andchronic inflammation, including atopic dermatitis and psoriasis. Inaddition, various neoplasms and tumor metastases can be treated.

[0186] Preferably, the pharmaceutical compositions are administeredintravenously. Thus, this invention provides compositions forintravenous administration which comprise a solution of the liposomessuspended in an acceptable carrier, preferably an aqueous carrier. Avariety of aqueous carriers can be used, e.g., water, buffered water,0.9% isotonic saline, and the like. These compositions can be sterilizedby conventional, well known sterilization techniques, or may be sterilefiltered. The resulting aqueous solutions may be packaged for use as isor lyophilized, the lyophilized preparation being combined with asterile aqueous solution prior to administration. The compositions maycontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents, wetting agents and the like, forexample, sodium acetate, sodium lactate, sodium chloride, potassiumchloride, calcium chloride, sorbitan monolaurate, triethanolamineoleate, etc.

[0187] The concentration of active ingredient in the pharmaceuticalformulations can vary widely, i.e., from less than about 0.05%, usuallyat or at least about 2-5% to as much as 10 to 30% by weight and will beselected primarily by fluid volumes, viscosities, etc., in accordancewith the particular mode of administration selected. For diagnosis, theamount of composition administered will depend upon the particular labelused (i.e., radiolabel, fluorescence label, and the like), the diseasestate being diagnosed and the judgment of the clinician.

[0188] The following examples serve to illustrate, but not to limit theinvention.

EXAMPLES I. Example I

[0189] A. General Overview

[0190] Distal cationic-poly(ethylene glycol)-lipid conjugates (CPL) weredesigned, synthesized and incorporated into conventional and stealthliposomes for enhancing cellular uptake. The present approach useseither inert, nontoxic or naturally occurred compounds as components forthe CPL synthesis. CPLs were synthesized with the followingarchitectural features: 1) a hydrophobic lipid anchor of DSPE forincorporating CPLs into liposomal bilayer; 2) a hydrophilic spacer ofpolyethylene glycol for linking the lipid anchor to the cationic headgroup; and 3) a naturally occurring amino acid (L-lysine) was used toproduce a protonizable cationic head group. The number of charged aminogroups can be controlled during the CPL synthesis. It has beendemonstrated that DSPE-CPLs were almost quantitatively incorporated intoliposomal bilayer by a hydration-extrusion method. Quite surprisingly,in an in vitro model, it was confirmed for the first time that liposomespossessing distal positively charged polymer conjugates with preferablyfour or more charges efficiently bind to host cell surfaces and enhancecellular uptake in mammalian cells.

[0191] B. Materials and Methods

[0192] 1. Abbreviations: DSPE,Distearoyl-sn-glycero-3-phosphoethanolamine; DSPC,1,2-distearoyl-sn-glycero-3-phosphocholine; DSPE-PEG₂₀₀₀,1,2-distearoyl-3-phosphatidylethanolamine-PEG₂₀₀₀; TFA, trifluoroaceticacid; CPL, cationic-poly (ethylene glycol)-lipid conjugate; DSPE-CPL,(cationic-polyethylene glycol)-DSPE conjugate; DSPE-CPL-1, DSPE-CPL withone positive charge; DSPE-CPL-2, DSPE-CPL with two positive charges;DSPE-CPL-4, DSPE-CPL with four positive charges; DSPE-CPL-8, DSPE-CPLwith eight positive charges; Rh-PE, (or Rho-PE),1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine Rhodamine Bsulfonyl).

[0193] 2. Chemical: t-Boc-NH-PEG₃₄₀₀-CO₂NHS was obtained from Shearwaterpolymers, Inc (Huntsville, Ala.). Nα, Nε-di-t-Boc-L-LysineN-Hydroxysuccinide Ester, triethylamine and cholesterol were obtainedfrom Sigma-Aldrich Canada Ltd (Oakville, ON). Trifluoroacetic acid,ethyl ether and chloroform were obtained from Fisher Scientific (FairLawn, N.J.). 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine and1,2-distearoyl-sn-glycero-3-phosphocholine were obtained from AvantiPolar Lipids, Inc (Alabaster, Ala.).1,2-distearoyl-3-phosphatidylethanolamine-PEG₂₀₀₀ was obtained fromGenzyme (Cambridge, Mass.).

[0194] 3. Synthesis of DSPE-CPL-1: To a solution of DSPE (121 mg, 161mmol) and Et₃N (200 μL) in CHCl₃ (2 mL) at 45° C. was addedt-Boc-NH-PEG₃₄₀₀-CO₂NHS (500 mg, 147 μmol in 2 mL dry CHCl₃), and thesolution was stirred for 3 hr at ambient temperature. The solution wasconcentrated under a nitrogen stream to dryness. The residue waspurified by repeat precipitation of the chloroform mixture solution withdiethyl ether until disappearance of DSPE spot on TLC. The purifiedDSPE-PEG conjugate was dissolved in 2 mL CHCl₃ followed by addition of 2mL TFA, and the reaction solution was stirred at room temperature for 4hr. The solution was again concentrated under a nitrogen stream todryness. The residue was purified by repeat precipitation of thechloroform mixture solution with diethyl ether to offer DSPE-PEG-NH₂ asDSPE-CPL-1 with one protonable cationic head group: yield 500 mg (120μmol, 80%); R_(f) 0.4 (CHCl₃/MeOH, 9/1, v/v); The ratio ofphosphoryl-lipid anchor and the distal primary amine was measured byphosphate and flourescamine assays and ¹H NMR.

[0195] 4. General procedure for the synthesis of DSPE-CPL-2. DSPE-CPL-4and DSPE-CPL-8 (see FIG. 2 for schematic): To a solution of DSPE-CPL-1(250 mg, 60 μmol) and Et₃N (200 μL) in CHCl₃ (2 mL) was added Nα,Nε-di-t-Boc-L-Lysine N-Hydroxysuccinide Ester (50 mg, 113 μmol in 2 mLdry CHCl₃), and the solution was stirred for 3 hr at ambienttemperature. Disappearance of positive amine-active spot on TLC bynihydrin visualization indicated that the reaction was completed. Thesolution was concentrated under a nitrogen stream to dryness. Theresidue was purified by repeat precipitation of the chloroform mixturesolution with diethyl ether until disappearance of t-Boc-Lysine spot onTLC. The purified DSPE-PEG-conjugates were dissolved in 2 mL CHCl₃followed by addition of 2 mL TFA, and the reaction solution was stirredat room temperature for 4 hr. The solution was again concentrated undera nitrogen stream to dryness. The residue was purified by repeatprecipitation of the chloroform mixture solution with diethyl ether tooffer DSPE-CPL2: yield 250 mg (57 μmol, 95%); R_(f) 0.4 (CHCl₃/MeOH,9/1, v/v); The ratio of phosphoryl-lipid anchor and the distal primaryamine was 1 measured by phosphate and flourescamine assays. DSPE-CPL4and DSPE-CPL8 were synthesized in a similar manner.

[0196] 5. Preparation of large unilamellar vesicles: Large unilamellarvesicles (LUV) were prepared by extrusion as described by Hope et al.(see Hope, M. J., et al., (1985) Production of large unilamellarvesicles by a rapid extrusion procedure. Characterization of sizedistribution, trapped volume and ability to maintain a membranepotential. Biochim. Biophys. Acta. 812, 55-65). Appropriate amounts oflipid mixtures (DSPC/Chol, 60:40 mol/mol) with or without DSPE-CPLs (asset out in Table 3) containing trace amounts of Rh-PE in chloroform,were dried under a stream of nitrogen gas to form a homogeneous lipidfilm. The trace amount of solvent was then removed under a vacuumovernight. The lipid film was hydrated in HBS buffer (pH 7.5) with orwithout HPTS (50 mM) by vortex mixing. The resulting multilamellarvesicles (MLVs) were extruded 10 times through two stacked 100 nmpolycarbonate filters (Nuclepore) employing an extrusion device (LipexBiomembranes, Inc., Vancouver, BC, Canada) at 65° C. UnincorporatedDSPE-CPLs and in some cases untrapped free HPTS were removed bychromatography using a 1.1×20 cm Sepharose CL-6B column (Sigma ChemicalCo., St. Louis, Mo., USA) equilibrated with HBS buffer.

[0197] 6. Determination of liposome size: Liposome size was determinedby quasi-elastic light scattering (QELS) using a Nicomp 370 submicronparticle sizer (Santa Barbara, Calif.).

[0198] C. Results and Discussion

[0199] This example was carried out to synthesize and assess theefficacy of the distal positively charged cationic polymer lipidconjugates (CPL) to enhance the cellular uptake of CPL-incorporatedliposomes. The present approach uses inert, nontoxic and naturallyoccurring compounds, e.g., amino acids, as components for the CPLsynthesis. Several CPLs were designed with the following architecturalfeatures: 1) a hydrophobic lipid anchor for incorporating the CPLs intothe liposomal bilayer; 2) a hydrophilic spacer for linking the lipidanchor to the cationic head group; and 3) a cationic head group.Moreover, the amount and nature of the cationic group can be changedaccording to the final application. In this example, a naturallyoccurring amino acid, L-lysine, was used to produce a protonizable aminogroup. The number of amino group can be controlled during the CPLsynthesis.

[0200] In analyzing these compounds, structure-function relationships inthese cellular uptake enhancers may be identified. As an initial step, avariety of these CPLs with differing amounts of charge were screened fortheir ability to enhance uptake (see, FIGS. 5-7). In addition, thephysico-chemical properties of the synthesized CPLs and the ability ofthese CPLs to incorporate into the liposome bilayers were also studied(see, Tables 1-3 and FIGS. 5-7). In an in vitro model, it was confirmedthat these distal charged polymer conjugates significantly enhanceliposome uptake in mammalian cells. TABLE 1 Physicochemical propertiesof cationic CPL Sample NH₂/P ratio DSPE-CPL-1 0.98 DSPE-CPL-2 2.05DSPE-CPL-4 3.96 DSPE-CPL-8 7.88

[0201] TABLE 2 pH gradients for CPL-liposomes Sample Lipid composition ΔpH 1 DSPC/Ch (60:40) 1.84 2 DSPC/Ch/CPL-4 (57.5:40:2.5) 1.11 3DSPC/Ch/CPL-8 (57.5:40:2.5) 0.85 4 DSPC/Ch/PEG-PE (54:40:6) 1.59 5DSPC/Ch/PEG-PE/CPL-4 (54:40:2:4) 1.01 6 DSPC/Ch/PEG-PE/CPL-8 (54:40:2:4)1.13

[0202] TABLE 3 CPL incorporated liposomes and their properties. Lipidcomposition Size (nm) CPL incorp. (%)  1, DSPC/Ch(60:40) 110 —  2,DSPC/Ch/CPL-1(57.5:40:2.5) 120 98.5  3, DSPC/Ch/CPL-2(57.5:40:2.5) 12294.5  4, DSPC/Ch/CPL-4(57.5:40:2.5) 122 98.1  5,DSPC/Ch/CPL-8(57.5:40:2.5) 122 97.6  6, DSPC/Ch/CPL-1(55:40:5) 120 98.5 7, DSPC/Ch/CPL-2(55:40:5) 122 94.5  8, DSPC/Ch/CPL-4(55:40:5) 122 98.1 9, DSPC/Ch/CPL-8(55:40:5) 122 97.6 10, DSPC/Ch/PEG-PE(54:40:6) 128 —11, DSPC/Ch/PEG-PE/CPL-1(54:40:2:4) 130 96.7 12,DSPC/Ch/PEG-PE/CPL-2(54:40:2:4) 130 101 13,DSPC/Ch/PEG-PE/CPL-4(54:40:2:4) 130 104 14,DSPC/Ch/PEG-PE/CPL-8(54:40:2:4) 130 110 15, DSPC/Ch(60:40) 110 — 16,DSPC/Ch/CPL-4(57.5:40:2.5) 120 98.5 17, DSPC/Ch/CPL-8(57.5:40:2.5) 12294.5 18, DSPC/Ch/PEG-PE(54:40:6) 128 — 19,DSPC/Ch/PEG-PE/CPL-4(54:40:2:4) 130 96.7 20,DSPC/Ch/PEG-PE/CPL-8(54:40:2:4) 130 101

II. Example II

[0203] This example illustrates that LUVs containing CPL₄ can be formedby a detergent dialysis method.

[0204] The LUVs contain DOPE, DODAC, PEG-Cer-C20, and CPL₄[3.4K] (orCPL₄[1K]). Two preparations were made with the CPL comprising 4 mol % ofthe original lipids. TABLE 4 Lipid mol-% DODAC 6 DOPE 79.5 CPL₄ 4 PC-C2010 Rho-PE 0.5

[0205] The lipids indicated above were co-dissolved in chloroform, whichwas then removed under nitrogen followed by 2 hours under high vacuum.The dry lipid mixture (10 μmol total) was then hydrated in 83 μL of 1 MOGP and 1 mL Hepes-buffered saline (20 mM Hepes 150 mM NaCl pH 7.5) at60° C. with vortexing until all the lipid was dissolved in the detergentsolution.

[0206] The lipid-detergent mixture was transferred to Slide-A-Lyzerdialysis cassettes, and dialysed against at least 2 L HBS for 48 hours,with a least two changes of buffer in that time. Removal of detergent bydialysis results in formation of LUVs. To determine whether all of theCPL was incorporated into the LUVs following dialysis, the lipid sampleswere fractionated on a column of Sepharose CL-4B (see FIGS. 8A and 8B).The fractionation profiles show LUVs formed with either CPL₄[3.4K] orCPL₄[1K].

[0207] The final concentration of CPL in the LUV fraction (fractions7-10) was estimated from initial and final dansyl/rhodamine ratios, andfrom estimating the proportion of total dansyl and rhodaminefluorescence present in the LUV peak.

[0208] Essentially identical results were obtained.

[0209] In order to examine the effect of increasing the initial CPLconcentration, a sample was made with the following proportions: TABLE 5Lipid mol-% DODAC 6 DOPE 71.5 CPL4 12 PC-C20 10 Rho-PE 0.5

[0210] The column profile for fractionation of this sample is shown inFIG. 8C.

[0211] The results for all 3 samples are given below: TABLE 6 originalfinal mol Sample mol % CPL %-inserted % CPL 4 64.6 2.6 4 82.8 3.3 1250.9 6.1

[0212] Conclusion: LUVs containing CPL₄ can be formed by detergentdialysis. Not all of the CPL₄ is incorporated into the vesicle, and theproportion that is incorporated falls as the initial CPL/lipid molarratio is increased. In the present case, beginning with 4 mol % CPL,about 3 mol % was incorporated into the LUV. For an initial CPL contentof 12 mol %, a final content of 6 mol % was achieved. It is also worthnoting that the behavior of the CPL₄[1K] is very similar to that of theCPL₄[3.4K]. This is also true in post-insertion studies. In certaininstances, the ideal length of the hydrophilic spacer will allow thecationic groups to extend out from the liposomal surface at a distanceshorter than the normal neutral PEG that is typically being used toprovide stealth properties for increased liposomal circulationlifetimes.

III. Example III

[0213] A. Overview

[0214] In this example, a non-specific targeting approach is describedthat involves increasing the electrostatic attraction between liposomesand cells by incorporation of positively-charged lipid molecules intopreformed vesicles. This approach leads to dramatic increases in cellbinding/uptake in vitro in BHK cells. The methodology is demonstrated towork for neutral vesicles and for vesicles composed of lipids used inthe construct of lipid-based gene carriers. The approach outlined hereinthus has numerous applications ranging from delivery of conventionaldrugs to gene therapy.

[0215] B. Materials and Methods

[0216] 1. Materials: 1,2-dioleoylphosphatidylcholine (DOPC),1,2-dioleoylphosphatidylethanolamine (DOPE), and1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(Lissamine Rhodamine BSulfonyl) (Rhodamine-PE) were obtained from Avanti Polar Lipids.Cholesterol was obtained from Sigma Chemical Co. DODAC and PEGCerC20,PEGCerC 14, and PEGCerC8 were generous gifts from Inex Pharmaceuticals.

[0217] 2. Synthesis of cationic-PEG lipids: The details of the synthesisof the CPLs is described herein. Two types of CPLs were synthesizedwhich differed in the lipid anchor portion of the molecule. In one, theanchor was distearoylglycerol (DSG), while the other containeddistearoylphosphatidylethanolamine (DSPE). The molecule consists of theanchor portion, to which is attached a PEG₃₄₀₀ chain. At the end of thePEG chain, a charged “headgroup” is attached, often made up of lysineresidues linked together. By modifying the headgroup region, CPLs weresynthesized which contained 1 (mono, or M), 2 (di, or D), 3 (tri, or T),and 4 (quad, or Q) positive charges. Several different Quad CPLs weresynthesized, hence these are numbered Q1 through Q5. The nomenclaturechosen to describe these compounds specifies the type of lipid anchorand the identity of the headgroup (e.g., d-DSPE-CPL-Q5). The lower case“d” indicates a dansylated derivative.

[0218] 3. Preparation of Vesicles by Detergent Dialysis. In general,vesicles were formed using a detergent dialysis method (see, Wheeler, J.J., et al. (1999) Stabilized plasmid-lipid particles: construction andcharacterization. Gene Therapy 6, 271-281, the teachings of which areincorporated herein by reference). The lipids, as described in ExampleII, were co-dissolved in chloroform in the appropriate ratios, followingwhich the chloroform was removed under a stream of nitrogen and placedunder high vacuum for 2 hours. An aliquot of the non-ionic detergentoctylglucopyranoside (1 M in water) (OGP) was then added to the drylipid film, which was incubated for 10-20 minutes at 60° C. withfrequent vortexing. This was followed by addition of 20 mM HEPES 150 mMNaCl pH 7.5, with further warming and vortexing until all the lipid wasdispersed and a clear solution was obtained. For 20 mg of lipid, 0.125mL of OGP and 1 mL of HBS were used. The lipid-detergent solutions (1-2mL) were then transferred to Slide-A-Lyzer dialysis membranes (3 mLvolume) and exhaustively dialysed at room temperature against HBS over aperiod of 48 hours. In general, a total volume of 8-10 L of HBS was used(4-5 changes of 2L) for sample volumes of 1-8 mL.

[0219] Vesicles of DOPC and DOPC/Chol (55:45) were prepared by extrusionas previously described (Hope, M. J., et al., (1985) Production of largeunilamellar vesicles by a rapid extrusion procedure. Characterization ofsize distribution, trapped volume and ability to maintain a membranepotential. Biochim. Biophys. Acta 812, 55-65).

[0220] 4. Insertion of Cationic PEG-lipids into preformed vesicles. Thecationic PEG-lipids (CPLs) were stored as micellar solutions in HBS or,in a few cases, in methanol. The CPL and the vesicles were combined togive the desired molar ratio (up to 11.6 mol % CPL relative to vesiclelipid), and incubated for a given time at the desired temperature. Formost insertions, the standard conditions involved a 3 hour incubation at60° C. Following insertion, the samples were cooled on ice, and theCPL-LUV was separated from free CPL by passage down a column (1.5×15 cm)of Sepharose CL-4B equilibrated in HBS. FIG. 16 illustrates theinsertion protocol for SPLPs (an analogous procedure).

[0221] The insertion levels of CPL were measured by fluorescence. In allcases, the vesicles contained either 0.25 mol % or 0.5 mol %rhodamine-PE, and the CPL contained a dansyl group. After combining theCPL and lipids, a 15 μL aliquot (initial fraction) was set aside foranalysis. The amount of CPL inserted into the vesicles could then bequantified by measuring the initial dansyl/rhodamine (D/R) fluorescenceratio, and the D/R ratio of the isolated CPL-LUVs. Fluorescenceparameters: for the rhodamine assay, the excitation wavelength was 560nm, and the emission wavelength was 590 nm. For the dansyl assay, theexcitation wavelength was 340 nm, and the emission wavelength was 510nm. In general, the excitation and emission slit widths were 10 and 20nm, respectively. The assay was performed as follows: to an aliquot ofthe initial sample (2-3 μL) or the CPL-LUV (20-40 μL) was added 30 μL of10% Triton X-100 followed by 2 mL of HBS. The fluorescence levels ofboth the dansyl and rhodamine labels were read consecutively using awavelength program as per the above parameters with an emission filterof 410 run. The %-insertion was calculated as follows:

%-insertion=([D/R] _(CPL-LUV))*100/[D/R] _(INITIAL)

[0222] 5. Measurement of Lipid Concentrations: Following insertion, itis necessary to know the lipid concentration of each sample for cellbinding studies. This can be done quickly by fluorescence. Followingdetergent dialysis, the lipid concentration of each sample was measuredusing the standard phosphate assay (Fiske, C. H., and Subbarow, Y.(1995) The colorimetric determination of phosphorus. J. Biol. Chem. 66,375-400). An aliquot was then diluted to approximately 3 mM. Bycomparing the rhodamine fluorescence of this sample, whose lipidconcentration is known, with the CPL-LUVs prepared from that stock,allows determination of CPL-LUV concentrations. Lipid concentrations ofLUVs were measured using the standard phosphate assay. Following CPLinsertion, lipid concentrations were estimated for cell binding studiesfrom the rhodamine fluorescence.

[0223] 6. Uptake of CPL-containing LUVs by BHK cells. Approximately 10⁵BHK cells were incubated in PBS/CMG medium with 20 nmol ofDOPE/DODAC/PEGCerC20 (84/6/10) LUVs containing either (1) no CPL, (2) 8%DSPE-CPL-D, (3) 7% DSPE-CPL-T1, or (4) 4% DSPE-CPL-Q5. Incubations wereperformed for 1, 2, 4, and 6 hours at 4° C. and 37° C., the formergiving an estimate of cell binding, and the latter of binding anduptake. By taking the difference of the two values, an estimate of lipiduptake at 37° C. was obtained. For each timepoint, the cells wereruptured and assayed for lipid and protein. Lipid concentrations weremeasured from rhodamine fluorescence, while protein was determined usingthe BCA assay. Lipid concentrations were measured using rhodaminefluorescence, while protein was determined using the BCA assay kitobtained from Pierce.

[0224] C. Results and Discussion

[0225] 1. Development of Insertion Protocol. The transfer of pegylatedlipids from micellar aggregates to vesicles has been previouslydescribed (see, Uster, P. S., et al., (1996) Insertion of poly(ethyleneglycol) derivatized phospholipid into pre-formed liposomes results inprolonged in vivo circulation time. FEBS Letters 386, 243-246; Zalipsky,S., et al., (1997) Poly(ethylene glycol)-grafted liposomes witholigopeptide or oligosaccharide ligands appended to the termini of thepolymer chains. Bioconjugate Chem. 8, 111-118). This idea was testedwith DSPE-CPL's. This is demonstrated in FIG. 9(A) for DOPC LUVs. Theco-elution of the dansyl and rhodamine labels demonstrates incorporationof the CPL in the LUVs. In this case, 84% of the CPL was incorporatedinto the LUVs, and thus only a trace of free CPL is observed trailingthe CPL-LUV fractions. This is more clearly seen in FIG. 8(B), where theDSPE-CPL-Q5 has been inserted into a more complex positively chargedvesicles composed of DOPE/DODAC/PEGCerC20 (84/6/10). Here, theco-elution of the two fluorescent labels at approx. 9 mLs demonstrates70% insertion of the CPL into the vesicles. The free CPL elutes in abroad peak centered at 16 mLs, which is separate from the vesicle peak,allowing for easy isolation of the CPL-LUV. Once inserted, theDSPE-CPL-Q5 is retained and does not exchange out of the vesicles. TheCPL-LUV fraction from FIG. 9(B) was re-eluted on the column of SepharoseCL-4B. As shown in FIG. 9(C), all of the CPL remains with the LUVs.

[0226] The effects of incubation temperature and time on the insertionprocess are shown in FIG. 10. DSPE-CPL-Q1 was incubated in the presenceof DOPE/DODAC/PEGCerC20 (84/6/10) at room temperature, 40° C., and 60°C., with aliquots withdrawn at 1, 3, and 6 hours. The highest insertionlevels were achieved at 60° C., which was therefore used in subsequentinsertions. Although slightly higher insertion was obtained at 6 hr, wechose 3 hr to minimize sample degradation.

[0227] Aside from time and temperature, the parameter that will have thegreatest influence on final CPL insertion levels is the initialCPL/lipid ratio. Assuming about 70% insertion, a series of incubationswere performed with CPL/lipid molar ratios varying between 0.011 to0.14, with the aim to achieve CPL-LUVs containing 1, 2, 4, 6, 8, and 12mol % CPL. These results are shown in FIG. 11, where it is seen that theinsertion level remains close to 70% up to an initial CPL/lipid ratio of0.095, above which it drops to 50% for CPL/lipid=0.14.

[0228] Similar results were obtained for other vesicle systems,including DOPE/DODAC/PEGCerC14 and DOPE/DODAC/PEGCerC8. In general, theinsertion levels obtained with DODAC-containing samples fell in therange of 70-80% for initial CPL/lipid <0.1. In order to see whether theinsertion levels were effected by the presence of cationic lipid,several experiments were performed on neutral vesicles containing DOPC.The compositions examined were: (1) DOPC, (2) DOPC/Chol, (3)DOPC/PEGCerC20, and (4) DOPC/Chol/PEGCerC20. The results, shown in FIG.12, reveal that for the DSPE-CPL-Q1, somewhat less insertion wasachieved in the neutral systems: between 45-65%. This may be due toreduced attraction between the negatively-charged DSPE anchor and themembrane surface. Regardless, the results demonstrate that significantinsertion can be achieved for both neutral and positive vesicles.

[0229] It should be noted that the insertion levels for the DSPE-CPL-Q5also shown in FIG. 13 are much higher than for the Q1 (70-84%). There isa reason for the differential behavior of the Q1 and Q5 CPLs in thesesystems, when in prior experiments they behaved very similar. Thisparticular batch of Q5 was prepared in methanol, a solvent in which thelipids may exhibit greater storage stability. As explained herein, ithas been found that the presence of methanol in the incubation mixtureleads to higher insertion.

[0230] A large number of insertions have been performed using other CPLsin addition to the Q5 and Q1. These results are summarized in Tables 7and 8, (FIGS. 23 and 24) where some composition-dependent trends can beascertained. First, the same trend seen above in FIG. 11 with the Q5hold for several CPLs with differing charge. As the initial ratio ofCPL/lipid is increased, the percentage of CPL inserted decreases. If welook at the T1, Q1, and Q5 incubations where CPL/lipid=0.022-0.024, the%-insertion ranges from 76-80%. However, for CPL/lipid=0.086-0.095, the%-insertion range decreases to 62-68%.

[0231] Another trend is illustrated in FIG. 13. For initial CPL/lipidratios >0.04, slightly less CPL-Q1 is inserted into LUVs containingPEG-Cer-C20 than into those containing either of the shorter chain PEGs.In addition to the type of PEG anchor present, the quantity of PEG-Ceralso has an effect on insertion, as seen in FIG. 14. As the PEG-Cer-C20content is increased from 4 to 10 mol %, the insertion levels of CPL-Q5fall from 71 to 62%.

[0232] As those of skill in the art will readily appreciate, the lipidanchor can be varied and the insertion levels may vary depending on thelipid used as the lipid anchor. For instance, some experiments wereperformed with CPL containing a DSG (distearoylglycerol) anchor: in allcases the insertion levels were much lower, from 17-40%, than in CPLcontaining a DSPE anchor. Using the methods and assays of the presentinvention, those of skill in the art can readily identify suitable lipidanchors.

[0233] In order to check for possible aggregation following CPLinsertion, quasi-elastic light scattering (QELS) was used to examine theeffect of insertion on particle diameter. DOPE/DODAC/PEG-Cer-C20vesicles were found to have a diameter of 119±39 nm. Following insertionof 1.8 mol % CPL4b, a slight increase in diameter to approx. 135±42 nmwas observed, but both the mean diameter and standard deviation remainedconstant up to 7 mol % CPL. The increase from 120 nm to 135 nm couldreflect a slightly larger diameter resulting from the presence of thelonger CPL PEG chains or it could indicate a small amount of vesicleaggregation. To differentiate between these two possibilities, CPL-LUVswere examined by fluorescence microscopy, using a rhodamine filter.While control LUVs exhibited no signs of aggregation, significant levelswere observed for CPL-LUVs. However, it was found that addition of 40 mMCaCl₂ completely prevented this effect.

[0234] As described in Materials & Methods, estimates were obtained forthe uptake of various CPL-LUVs on BHK cells incubated on PBS/CMG. Thedata, shown in FIG. 15, reveals that the presence of positive charge onthe CPLs can lead to significant enhancement in uptake by BHK cells.LUVs composed of DOPE/DODAC/PEGCerC20 (and thus exhibiting a netpositive charge) showed little uptake on the BHK cells. LUVs containing8 mol % of DSPE-CPL-D showed similar low uptake values. Uptake was onlyslightly increased by the presence of 7 mol % of DSPE-CPL-T1. However, asignificant increase in uptake was realized for DSPE-CPL-Q5 present atonly 4.1 mol % Several points can be surmised from this data. While itis clear that an increase in the positive charge present at somedistance from the LUV surface leads to an increase in uptake, it is nottotal charge alone that plays a role in enhanced cell binding. Thequantity of positive charge present for the DSPE-CPL-D and DSPE-CPL-Q5samples is approximately equal, and yet the former shows little bindingcompared to the latter. The DSPE-CPL-T1 sample has a greater positivecharge than the DSPE-CPL-Q5 sample, and yet exhibits only ⅓ the uptake.It would appear that localization of a sufficient positive chargedensity at the distal end of the CPL molecule is an important parameterin ensuring interaction with cells. In a preferred embodiment, at leastfour charges are used to achieve efficient cell binding.

[0235] The dramatic effect of CPL insertion on LUV binding to BHK cellsis most clearly visualized using fluorescence microscopy. In the absenceof CPL, vesicles composed of DOPE/DODAC/PEG-Cer-C20 and containing atrace of rhodamine-PE exhibit little binding to cells. Incorporation of3 mol % CPL4b leads to high levels of vesicle binding and uptake.Although much of the lipid appears to be binding to the cell surface,some small punctate structures can be seen, indicating that uptake ofvesicles is also occurring. An important point to note is that the cellsappear healthy following incubation in the presence of the CPL-LUVs. Incontrast, DNA-cationic lipid complexes are known to display significanttoxicity.

[0236] One of the major remaining hurdles in liposomal drug delivery isthe problem of how to ensure that the contents of a carrier system aretaken up and utilized by a specific target cell. It is now believed thatthe cellular uptake of liposomes involves adsorption or binding at thecell surface, followed by endocytosis. Thus factors which interfere withcellular binding will lead to low levels of intracellular delivery. Thisis of particular importance for ‘stealth’ or long-circulating liposomesthat are coated with a surface layer of a hydrophilic polymer such asPEG. The very characteristic of the PEG coating which impartslong-circulation lifetimes—the formation of a steric barrier thatprevents interaction with serum proteins, will also minimizeinteractions with cells. On the other hand, factors that enhance surfacebinding may be expected to lead to increased cellular uptake. Oneapproach involves attaching molecules specific for membrane receptors toliposomal surfaces. Possible candidates include oligopeptides (see,Zalipsky et al., Bioconjugate Chemistry 6, 705-708 (1995); Zalipsky etal., Bioconjugate Chemistry 8, 111-118 (1997)) oligosaccharides (see,Zalipsky et al., Bioconjugate Chemistry 8, 111-118 (1997)), folate (see,Gabizon et al., Bioconjugate Chemistry 10, 289-298 (1999); Lee et al.,Journal of Biological Chemistry 269, 3198-3204 (1994); Reddy et al.,Critical Reviews in Therapeutic Drug Carrier Systems 15, 587-627 (1998);Wang et al., Journal of Controlled Release, 53, 39-48 (1998)),riboflavin (see, Holladay et al., Biochimica et Biophysica Acta 1426,195-204 (1999)), or antibodies (see, Meyer et al., Journal of BiologicalChemistry 273, 15621-15627 (1998); Kao et al., Cancer Gene Therapy 3250-256 (1996); Hansen et al., Biochimica et Biophysica Acta 1239,133-144 (1995)]. An alternate approach is to modify the chargecharacteristics of the liposome. It is well known that inclusion ofeither negative (see, Miller et al., Biochemistry 37, 12875-12883(1998); Allen et al., Biochimica et Biophysica Acta 1061, 56-64 (1991);Lee et al., Biochemistry 32, 889-899 (1993); Lee et al., Biochimica etBiophysica Acta 1103, 185-197 (1992)) or positive (see, Miller et al.,Biochemistry 37, 12875-12883 (1998)) charges in liposomes can lead toenhanced cellular uptake. Cationic DNA-lipid complexes, which areefficient in vitro transfection agents (see, Felgner et al. Nature 337,387-388 (1989); Felgner et al., Proceedings of the National Academy ofSciences of the United States of America 84, 7413-7417 (1987); Kao etal., Cancer Gene Therapy 3 250-256 (1996); Felgner et al., Annals of theNew York Academy of Sciences 772, 126-139 (1995); Jarnagin et al.,Nucleic Acids Research 20, 4205-4211 (1992)), are taken up viaendocytosis.

[0237] This example describes a new approach for enhancing theinteraction of liposomes with cells, a necessary step in the developmentof non-viral systems capable of intracellular delivery. The approachinvolves the insertion of novel cationic-PEG-lipids into pre-formedliposomes, leading to a cationic vesicle in which the positive chargeinvolved in cell interaction is located some distance away from thevesicle surface. The process is illustrated in FIG. 16 for the insertionof a CPL₄ into sterically-stabilized LUVs composed of DOPE, the cationiclipid DODAC, and PEG-Cer-C20. This lipid composition was chosen forstudy for two reasons: first, it allows for efficient entrapment ofplasmid DNA within small vesicular structures by virtue of the presenceof positively charged DODAC (see, Wheeler et al. Gene Therapy 6, 271-281(1999)), and thus has potential as a gene delivery system (see below).Secondly, this composition is representative of the manysterically-stabilized drug delivery systems which contain PEG-lipids.Insertion of CPLs leads to localization of positive charge above thesurface PEG layer, thereby allowing electrostatic interactions betweenthe CPLs and cell surfaces. This should lead to increased cellularinteractions for both conventional- and PEG-containing liposomes.

[0238] The CPLs are conjugates of DSPE, a dansyl-lysine moiety, thehydrophilic polymer PEG₃₄₀₀, and a mono- or multivalent cationicheadgroup. The PEG functions as a spacer, separating the chargedheadgroup from the lipid anchor and vesicle surface. Incubation of awide variety of neutral and cationic LUVs with micellar CPLs resulted inthe incorporation of up to 6-7 mol % (relative to total vesicle lipid)of CPL in the outer vesicle monolayer (see tables in FIGS. 23 and 24).The insertion efficiency was quite high, with approximately 70-80% ofadded CPL incorporating into the LUVs (see tables in FIGS. 23 and 24).The most important factors influencing the CPL insertion levels were theincubation temperature (FIG. 10) and initial CPL/lipid ratio (FIG. 11).The composition of the liposome was found to affect the final CPL levelsto a lesser degree (see tables in FIGS. 23 and 24). Following insertion,the CPL-LUV could be efficiently separated from free CPL by gelexclusion chromatography. Similar insertion levels were obtained for allCPLs, with headgroup charges ranging from one to four charges permolecule. With this knowledge, vesicles could be prepared containing adesired level of CPL with reasonable accuracy.

[0239] High insertion levels (up to 7 mol %) could be achieved forvesicles containing as much as 10 mol % PEG-Cer-C20. It is possible thata portion of the PEG-Cer's are lost during the insertion process, asPEG-Cer's will exchange from vesicles during circulation. This mayexplain why the highest insertion levels are achieved with PEG-Cer-C8,which has the greatest propensity to exchange. However, analysis of LUVsand SPLPs containing PEG-Cer-C20 by HPLC before and after insertion ofCPL4 reveal only a slight loss of PEG-Cer-C20 (from about 10 mol % to 8mol %).

[0240] As shown in FIG. 15, cationic LUVs composed ofDOPE/DODAC/PEG-Cer-C20 exhibit little uptake when incubated on BHKcells. Although positively charged vesicles exhibit enhanced binding tosome cell lines, this can be attenuated by the presence of PEG on theliposome surface (see, Miller et al., Biochemistry 37, 12875-12883(1998)). Clearly, for these systems, the presence of 6 mol % ofpositively charged DODAC leads to only low uptake levels after 6 hours.Incorporation of approximately 7 mol % of dicationic-CPL has littleeffect on uptake, which was only slightly improved in the presence ofapprox. 7 mol % tricationic-CPL. The best results were obtained with theCPL4b (at 4 mol %), which possessed 4 positive charges. At 6 hoursincubation, a ten-fold increase in uptake was observed relative to thestarting vesicles. Several points can be surmised from this data. Thefirst is that the presence of positively charged groups at some distancefrom the LUV surface can lead to significant increases in cellularuptake. In this case, the positive charges of the CPL (PEG MW=3400) arelocated above the surface coating of PEG (MW=2000), and thus areavailable for interactions with cells. However, it is not total chargealone that plays a role in enhanced cell binding. The quantity ofpositive charge present for the CPL₂ and CPL₄b samples is approximatelyequal, and yet the former shows little uptake compared to the latter.The CPL₃ sample has a greater positive charge than the CPL₄b sample, andyet exhibits only ⅓ the uptake. It would appear that localization of asufficient positive charge density at the distal end of the CPL moleculeis an important parameter in ensuring interaction with cells. At leastfour charges seem to be required for efficient cell binding to occur.

[0241] The protocol described for insertion of CPL into conventional andsterically-stabilized CPL is ideal for demonstrating the methodologyusing in vitro applications. In both cases, the added positive charge isphysically distant from the surface, and is available for interactionswith cells. This is particularly important for polymer-coated vesiclesthat are designed for minimal interactions with serum proteins and cellssuch as macrophages. However, this system may not be ideal for in vivoapplications, where it may be desirable to initially hide or screen theCPL charge to reduce clearance and allow accumulation of the vesicles atthe tissue of choice. Thus, alternative embodiments employ shorter PEGspacer chains in the CPL, or longer PEG chains in the PEG-Cer molecules.The PEG-Cer molecules are known to exchange out of the particle duringcirculation see, Webb et al., Biochimica et Biophysica Acta 1372,272-282 (1998)], which would leave the CPL exposed for cellularinteractions.

[0242] As mentioned above, the cationic liposomes employed in thepresent study are composed of a fusogenic lipid (DOPE), a cationic lipid(DODAC), and a stabilizing lipid (PEG-Cer-C20), the latter of whichimparts long-circulating properties to the vesicles. This lipidcomposition was modeled after a new class of lipid-based DNA carriersystems known as stabilized plasmid-lipid particles (SPLPs) see, Wheeleret al. Gene Therapy 6, 271-281 (1999)). SPLPs are small (70 nm)particles that encapsulate a single plasmid molecule. The presence of aPEG coating on the liposome surface imparts long-circulation propertiesas well as protecting the plasmid from degradation by serum nucleases.SPLPs thus represent the first carrier systems with real potential forsystemic in vivo gene therapy applications. The approach described heregreatly enhances the tranfection potency of these particles byincreasing cellular binding and uptake, which leads to increasedintracellular delivery of plasmids. The inclusion of CPL in conventionalformulations (e.g., anticancer drugs) also leads to increased efficacy.

IV. Example IV

[0243] A. Overview

[0244] This example employs CPLs incorporated into stable plasmid-lipidarticles (SPLPs) for in vitro transfection of cells.

[0245] Incubation of these particles on BHK cells for up to 8 hoursresulted in an increase in uptake as the amount of inserted CPLincreased from 2-4 mol %. Transfection of the SPLP system increased withthe addition of CPL with 15 mM CaCl₂ in the transfection media. The SPLPalone showed very low transfection at both a 4 and a 9 hour transfectionfollowed by 24 hour complete incubation in fresh media. The addition of15 mM CaCl₂ final concentration in the media to the SPLPs, increasedtransfection on BHK cells by 10-fold at both time points. In thepresence of 15 mM CaCl₂, SPLP+2%, 3% and 4% CPL transfect 2000- to5000-times higher than that of the SPLP alone at both time points. The 4mol % CPL shows the greatest increase in transfection: approximately4500 times higher, followed by the 3% and then the 2% CPL samples.Therefore, the presence of the CPL, DSPE-Quad5 in the SPLP increased inboth uptake and transfection to levels comparable to or above thoseachieved with the complexes.

[0246] B. Materials and Methods

[0247] 1. Synthesis of the DSPE-Quad5: The dansylated DSPE-Quad5 (CPL)was prepared in our laboratory as described by Chen et al (2000).

[0248] 2. Incorporation of DSPE-Quad5 Into SPLP: Inex Pharmaceuticals,Inc. supplied the SPLP. The incorporation of the CPL into the SPLP wasperformed by incubation of the CPL with the SPLP at 60° C. for 2-3 hoursin HBS. The resulting mixture was then passed down a Sepharose CL-4Bcolumn equilibrated with HBS, 75 mM CaCl₂, pH 7.5 to remove theunincorporated CPL from the SPLP with the incorporated CPL. Fractions (1mL) were collected and assayed for CPL (dansyl assay), phospholipid, andDNA (PicoGreen assay). The final samples were prepared to contain 2, 3,or 4 mol % of the CPL. The dansyl assay involved preparing a standardcurve of 0.5 to 2.5 mol % of dansylated CPL in BBS and determining theconcentration of the CPL in the sample. The phospholipid was extractedfrom the SPLP by extracting the lipid using the Bligh-Dyer extractiontechnique (Bligh & Dyer, 1952) and then performing a Fiske-Subarrowassay on the organic phase of the extraction. The PicoGreen assay wasperformed by comparing the sample in the presence of PicoGreen andTriton X-100 using a DNA standard curve. The final % insertion of theCPL was determined by dividing the CPL concentration by the lipidconcentration.

[0249] The optimal time for insertion of the CPL into the SPLP wasdetermined using SPLP prepared with 0.5 mol % Rh-DSPE. 15 nmol of thedansylated CPL (DSPE-Quad5) was mixed with 200 nmol of the labeled SPLPand the sample was incubated at 60° C. for various time points (0.5, 1,2, 3, and 4 hours). At these time points the sample was removed from thewater bath and was passed down a Sepharose CL-4B column. The majorfraction was collected from the column and the dansyl to rhodaminefluorescence ratios were measured. The parameters used for the rhodaminefluorescence were a λ_(ex) of 560 nm and a λ_(em) of 600 nm and for thedansyl fluorescence were a λ_(x) of 340 m and a λ_(em) of 510 nm. Theexcitation and emission slit widths for both of these were 10 nm and 20nm, respectively. By comparison of the dansyl/rhodamine ratio for thesample before the column to that after the column, the % insertion wasdetermined at each time point.

[0250] 3. QELS of CPL-SPLP: The diameter of these particles wasdetermined using a Nicomp Particle Sizer.

[0251] 4. Freeze-Fracture EM Freeze-fracture EM was performed on the 2%,3%, and 4% CPL samples by methods which will be described by K. Wong

[0252] 5. Serum Stability of Particles: The stability of the DNA withinthese CPL-SPLP was determined by incubating the samples (25 μL),containing 6 μg of plasmid DNA (pLuc) for various time periods (0, 1, 2,and 4 hours) in 50% mouse serum (25 μL) at 37° C. At each time point,other than the zero time point, 11 μL of the mixture was removed, thevolume was made up to 45 μL using water and the samples were placed onice. The DNA was then extracted from the lipid using one volume ofphenol:chloroform (1:1). Following a 20 min centrifugation in amicrofuge, the top aqueous phase was removed. The zero time point wasobtained by removing 5.5 μL of the sample prior to serum addition andperforming the extraction. Twenty microliters of the aqueous phase wasthen mixed with 2 μL of loading buffer and the sample was run on a 1%agarose gel in TAE buffer. Following one hour, the gel was placed on atransilluminator and a photograph was taken.

[0253] 6. Lipid Uptake Studies: For the uptake studies, 1×10⁵ BHK cellswere grown on 12 well plates overnight in 2 mL of complete media(DMEM+10% FBS) at 37° C. in 5% CO₂. Then 20 nmol of the 2, 3, and 4 mol% CPL-SPLP samples containing 0.5% rhodamine-DSPE were mixed with HBS+75mM CaCl₂ to a final volume of 200 μL and this was added to the top ofthe cells followed by the addition of 800 μL of complete media. This wasallowed to incubate on top of the cells for 2, 4, 6, and 8 hours atwhich time the cells were washed three times with PBS and were lysedwith 600 μL of 0.1% Triton X-100 in PBS, pH 8.0. The rhodaminefluorescence of the lysate was then measured on a fluorometer using aλ_(ex) of 560 nm and a λ_(em) of 600 nm using slit widths of 10 and 20nm, respectively. An emission filter of 430 nm was also used. A 1.0 mLmicrocuvette was used. The lipid uptake was determined by comparison ofthe fluorescence to that of a lipid standard (5 nmol). This value wasthen normalized to the amount of cells present by measuring the proteinin 50 μL of the lysate using the BCA assay.

[0254] Fluorescence micrographs were taken on a Zeiss fluorescencemicroscope.

[0255] 7. Transfection Studies: For the in vitro transfection studies,5×10⁴ BHK cells were plated in 24-well plates in complete media. Thesewere incubated overnight at 37° C. in 5% CO₂. SPLP, SPLP+75 mM CaCl₂,DOPE:DODAC (1:1)/DNA complexes, and CPL-SPLP systems (2, 3, and 4 mol %CPL) containing 2.5 μg of DNA were made up to 100 μL using HBS or HBS+75mM CaCl₂ and were placed on the cells. Then 400 μL of complete media wasadded to this. At 4 and 9 hours, the transfection media was removed andreplaced with complete media containing penicillin and streptomycin fora complete 24 hour transfection. At the end of the transfection period,the cells were lysed with lysis buffer containing Triton X-100.Following this lysis, 10-20 μL of the lysated was transferred to a96-well luminescence plate. The luminescence of the samples on the platewere measured using a Luciferase reaction kit and a plate luminometer.The luciferase activity was determined by using a luciferase standardcurve and was normalized for the number of cells by measuring theprotein with the BCA assay on 10-20 μL of the lysated.

[0256] C. Results and Discussion

[0257]FIGS. 18A and B show that the uptake and transfection of the SPLPsystem is on the order of 105 times lower than complexes.

[0258] The CPL, DSPE-Quad5, will be used in the following studies. Itsstructure is shown in FIG. 16A. This molecule possesses four positivecharges at the end of a PEG₃₄₀₀ molecule, which has been covalentlyattached to the lipid DSPE. The incorporation of this CPL into emptyliposomes of the same composition as the SPLP has been describedpreviously in the above examples.

[0259] The incorporation of the CPL into the SPLP involves only a fewsteps. These steps are shown in FIG. 16B.

[0260] The DSPE-Quad5 was incorporated into SPLPs containingDOPE:PEG-CerC20:DODAC (84:10:6) at various concentrations of the CPL(from 2-4 mol %). The incorporation efficiencies for the various CPLpercentages were between 70 and 80% of the initial. In order to separatethe SPLPs possessing the CPL from the unincorporated CPL, gel filtrationchromatography was employed. A typical column profile for the 3%DSPE-Quad5 is shown in FIG. 19A. The CPL, lipid, and DNA all eluted fromthe column at the same time in a single peak. There was however a smallamount of unincorporated CPL that eluted at a later stage. To show thatthe incorporated CPL remains incorporated, the sample is re-eluted fromthe column (FIG. 19A). As it can be seen in FIG. 199B, no CPL is elutedin the later fractions of the column indicating that the CPL remainsassociated with the lipid.

[0261] To determine the optimal incubation period for the insertion ofthe CPL, a time course at 60° C. was performed (FIG. 20). From thisfigure, it can be determined that the optimal insertion occurs between 2and 3 hours.

[0262] The diameter of these particles containing the CPL was determinedby QELS to be 125 nm compared to the SPLP, which had a diameter of 109nm. To observe the structure of these particles compared to the SPLP inthe absence of the CPL, freeze-fracture EM was performed.

[0263] The serum stability of the SPLP in the presence and absence ofvarious amounts of the CPL was assayed (data not shown). Incubating freeDNA with 50% mouse serum for only 1 hour results in its completedegradation. The serum stability of the CPL-SPLPs was similar to thatfor the SPLP system. This indicates that the DNA in the CPL-SPLP is asprotected as that in the SPLP system without CPL.

[0264] The major objective of this study is to increase both the uptakeand transfection of the SPLP system using CPLS. FIG. 21 shows the timecourse for the uptake of rhodamine labeled SPLP in the presence (2, 3,or 4 mol %) and absence of the DSPE-Quad5 (0%). The uptake of the 4%system is higher than the 3% system, which is higher than the 2% system,and all three are much higher than the system without CPL. FIG. 22 shows4 h and 9 h time points of the same formulations.

V. Example V

[0265] This example illustrates the incorporation of a CPL into aStabilized Antisense-Lipid Particle (“SALP”).

[0266] A. Materials and Results

[0267] Distearoylphosphatidylcholine (DSPC), was purchased from NorthernLipids (Vancouver, Canada). 1,2-dioleoyloxy-3-dimethylammoniumpropane(DODAP or AL-I) was synthesized by Dr. Steven Ansell (InexPharmaceuticals) or, alternatively, was purchased from Avanti PolarLipids. Cholesterol was purchased from Sigma Chemical Company (St.Louis, Mo., USA). PEG-ceramides were synthesized by Dr. Zhao Wang atInex Pharmaceuticals Corp. using procedures described in PCT WO96/40964, incorporated herein by reference. [³H] or [¹⁴C]-CHE waspurchased from NEN (Boston, Mass., USA). All lipids were >99% pure.Ethanol (95%), methanol, chloroform, citric acid, HEPES and NaCl wereall purchased from commercial suppliers. Lipid stock solutions wereprepared in 95% ethanol at 20 mg/mL (PEG-Ceramides were prepared at 50mg/mL).

[0268] SALPs are first prepared according to the methods set out in PCTPatent Application No. WO 98/51278, published 19 Nov. 1998, andincorporated herein by reference. See also, J. J. Wheeler et al.,(1999), Gene Therapy, 6, 271-281. Briefly, a 16mer of[3H]-phosphorothioate oligodeoxynucleotide Inx-6295 (human c-myc) havingsequence 5′ T AAC GTT GAG GGG CAT 3′ (SEQ ID. No: 1) (in 300 mM citratebuffer, pH 3.80) was warmed to 65° C. and the lipids (in ethanol) wereslowly added, mixing constantly (DSPC:CHOL:DODAP:PEG-CerC14;25:45:20:10, molar ratio). The resulting volume of the mixture was 1.0mL and contained 13 mmol total lipid, 2 mg of antisenseoligodeoxynucleotide, and 38% ethanol, vol/vol. The antisense-lipidmixture was subjected to 5 cycles of freezing (liquid nitrogen) andthawing (65° C.), and subsequently was passed 10× through three stacked100 nm filters (Poretics) using a pressurized extruder apparatus with athermobarrel attachment (Lipex Biomembranes). The temperature andpressure during extrusion were 65° C. and 300-400 psi (nitrogen),respectively. The extruded preparation was diluted with 1.0 mL of 300 mMcitric acid, pH 3.8, reducing the ethanol content to 20%. The extrudedsample was dialyzed (12000-14000 MW cutoff; SpectraPor) against severalliters of 300 mM citrate buffer, pH 3.8 for 3-4 hours to remove theexcess ethanol. The sample was subsequently dialyzed againstHEPES-buffered saline (HBS), pH 7.5, for 12-18 hours to neutralize theDODAP and release any antisense that was associated with the surface ofthe vesicles. Encapsulation was assessed either by analyzing thepre-column and post-column ratios of [³H]-antisense and [¹⁴C]-lipid orby determining the total pre-column and post-column [³H]-antisense and[¹⁴C]-lipid radioactivity.

[0269] CPL is incorporated after the SALPs are prepared. Approximately 5μmol SALP were mixed with 3-10 mol % CPL (i.e., 0.15-0.5 μmol CPL). CPLwere stored as micellar solutions in HBS, or in methanol. When CPL wasadded in methanol, the final methanol concentration of 3-4%. Themixtures were incubated overnight at room temperature or at 40° C.Unincorporated CPL was removed from the SALP preparation by columnseparation (Sepharose CL-4B equilibrated with HBS, 75 mM CaCL₂ at pH7.5). Incorporation efficiency was between 34 and 60%. It is anticipatedthat other organic solvents may improve incorporation efficiency.

VI. Example VI

[0270] A. General Overview

[0271] In the present example, distal positively charged cationicpoly(ethylene glycol) lipid conjugates (CPL) were synthesized andassessed for their efficacy at enhancing the cellular uptake ofCPL-incorporated liposomes. It was confirmed that distal charged polymerconjugates bound to a liposome surface enhanced liposome uptake inmammalian cells in vitro.

[0272] B. Methods

[0273] Determination of the Critical Micelle Concentration (CMC)

[0274] The CMCs of the CPLs were determined using the NPN assay aspreviously reported by Brito and Vaz (see, Brito, R. M. M., and Vaz, W.L. C. (1986) Determination of the critical micelle concentration ofsurfactants using the fluorescent probe N-phenyl-1-naphthylamine. Anal.Biochem. 152, 250-255.). A series of different concentrations of CPLswere prepared in HBS buffer (25 mM Hepes, 150 mM NaCl, pH 7.4). 5 μM ofNPN (from a stock NPN solution in 95% ethanol) was added into the aboveCPL solutions. After incubation of the mixtures at room temperature for30 min, the fluorescence intensities at λ_(em)=410 nm using λ_(ex)=356nm on a Perkin Elmer LS 50 Luminescence Spectrometer.

[0275] C. Results

[0276] Uptake Enhancement of CPL-L UVs in Vitro. Cellular Uptake ofConventional CPL-Liposomes.

[0277] The in vitro cellular uptake of CPL-containing liposomes wasstudied on baby hamster kidney (BHK) cells. The liposome-associatedfluorescent lipid marker (Rh-PE) was used as a marker for lipid uptake.As shown in FIGS. 26 and 27, CPL₄ significantly enhances the cellularuptake compared to control samples (no CPL) using both PBS-CMG and serumcontaining medium. The time dependent uptake of CPL-LUVs reaches amaximum after 3 hr. FIG. 6 summarized the cell uptake of the differentCPL-containing vesicles after a four hour incubation. Compared to acontrol, reduced cell uptake was observed for CPL₁, a moderate increasefor CPL₂ (2 fold), and a large increase for both CPL₄ and CPL₈. Thesimilar degree of increase resulting from CPL₄ and CPL₈ indicates acharge density of four in the CPLs satisfies the requirement for maximumenhanced cellular uptake.

VII. Example VII

[0278] A. General Overview

[0279] This experiment describes the synthesis of a new class ofcationic lipids designed to enhance non-specific targeting by increasingthe electrostatic attraction between liposomes and cells.

[0280] B. Materials and Reagents

[0281] tBoc-NH-PEG₃₄₀₀-CO₂—NHS was obtained from Shearwater Polymers(Huntsville, Ala.). N_(α),N_(ε)-di-tBoc-L-lysine-N-hydroxysuccinimideester, N_(ε)-dansyl-L-lysine, N-hydroxysuccinimide (NHS), andN,N′-dicyclohexyl-carbodiimide (DCC) were purchased from Sigma-AldrichCanada (Oakville, ON). 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC)and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) were obtainedfrom Northern Lipids (Vancouver, BC). Fluorescamine and Rhodamine-DSPE(Rh-PE) were obtained from Molecular Probes (Eugene, Oreg.). Cholesterol(Chol) was obtained from Sigma Aldrich Canada (Oakville, ON).Trifluoroacetic acid, diethyl ether, methanol, triethylamine, andchloroform were obtained from Fisher Scientific (Vancouver, BC). Allreagents were used without further purification.

[0282] 1. General Methods

[0283] All reactions were performed in 16×100 mm glass test tubes. ¹HNMR spectra were obtained employing a Bruker MSL 200 spectrometeroperating at 200 MHz. Deuterated chloroform (CDCl₃) was used as thesolvent in the NMR experiments. Proton chemical shifts (δ) werereferenced to CHCl₃ set at 7.24 ppm. When signals were reasonablyresolved, their intensities were integrated to allow an estimation ofthe number of protons. The chemical shifts of exchangeable amino groupprotons, observed between 7-8 ppm, are not given. These peaks wereassigned on the basis of their removal by a D₂O exchange.

[0284] Phosphorus and fluorescamine assays were performed to confirm theratio of primary amine per phosphate in each CPL as follows.

[0285] The phosphate concentration of the CPL was determined using theFiske-Subarrow phosphorus assay (see, Fiske, C. H., and Subbarow, Y.(1995) The colorimetric determination of phosphorous. J. Biol. Chem. 66,375-400.). The primary amine concentration in the CPL was determinedusing the fluorophore, fluorescamine. A fluorescamine solution (0.6mg/mL) in acetone was prepared. An aliquot of CPL solution in HBS (2-4μL) was made up to 250 μL with 200 mM sodium borate, pH 8.0. To thismixture, 50 L of the fluorescamine solution was added dropwise withvortexing, followed by 1700 μL of water. The fluorescence of thissolution was measured using a Perkin-Elmer LS50 LuminescenceSpectrometer with λ_(ex), of 397 nm and λ_(em) of 475 nm, and excitationand emission slit widths of 10 nm. The primary amine concentration ofthe CPL was determined from a lysine standard curve.

[0286] tBoc-NH-PEG₃₄₀₀-CO₂—(N_(ε)-dansyl)lysine (1).tBoc-NH-PEG₃₄₀₀-CO₂—NHS (500 mg, 147 μmol) in 3 mL of dry chloroform wasadded slowly to a solution of N_(ε)-dansyl-L-lysine (65 mg, 171 μmol) in1 mL of methanol and 200 μL of triethylamine. After the reaction mixturewas stirred at room temperature for 3 h, the solvent was removed under aN₂ stream and further dried under vacuum. The crude product was washedby first dissolving it in a minimum amount of chloroform with warmingand then precipitating it out with the addition of 10 mL of diethylether. The ether was added while vortexing. Precipitation of 1 wasaccelerated by cooling. The precipitate was then pelleted bycentrifugation and the ether was discarded. This chloroform/ether washand precipitation procedure was repeated. The dry solid was thendissolved in 4 mL of chloroform and cooled in an ice bath for 15 min.Methanol (2 mL) was added if this cooled solution was clear. If aprecipitate (excess dansyl lysine) developed, it was filtered off priorto the addition of methanol. The chloroform/methanol solution was washedwith 1.2 mL of 0.1 M HCl. The chloroform phase was extracted, dried, andthe solid redissolved in 6 mL of chloroform/methanol (2:1 v/v) andwashed with 1.2 mL of distilled water. The chloroform phase wasextracted, dried to a thick paste andtBoc-NH-PEG-CO₂—(N_(ε)-dansyl)lysine (1) was precipitated with 10 mL ofether. After centrifugation and the removal of ether, the dried productis a light yellow solid. Yield: 520 mg (93%). TLC (silica gel)chloroform/methanol (85:15 v/v): R_(f) 0.56. ¹H NMR (CDCl₃): δ 1.08 (t),1.40 (s, 11H), 2.66 (s, 1H), 2.86 (s, 8H), 3.27 (q), 3.50 (t), 3.60 (s,309H), 3.96 (t), 4.19 (m[broad]), 5.03 (s[broad], 1H), 5.22 (t, 1H),5.43 (d, 1H), 7.16 (d, 1H), 7.51 (q, 2H), 8.19 (d, 1H), 8.27 (d), 8.50(d, 1H) ppm.

[0287] Dansylated CPL₁-tBoc (3). First,tBoc-NH-PEG₃₄₀₀-CO₂—(N_(ε)-dansyl)lysine-NHS (2) was prepared asfollows. A solution of tBoc-NH-PEG₃₄₀₀-CO₂—(N_(ε)-dansyl)lysine (1) (500mg, 132 μmol) and NHS (31.5 mg, 274 μmol) in 2 mL of dry chloroform wasadded to DCC (42.8 mg, 207 μmol) dissolved in 1 mL of dry chloroform.The reaction mixture was stirred for 2 h at room temperature. Theby-product, dicyclohexyl urea (DCU), was filtered using a Pasteurpipette with a cotton plug. The filtrate, containingtBoc-NH-PEG₃₄₀₀-CO₂—(N_(ε)-dansyl)lysine-NHS (2), was slowly added to asolution of DSPE (120.6 mg, 161 μmol) in 2 mL of dry chloroform and 200μL of triethylamine. The dissolution of DSPE in dry chloroform andtriethylamine required warming to 65° C. After the reaction mixture wasstirred at room temperature for 3 h, it was dried, and chloroform/etherwashed and precipitated as described earlier until the disappearance ofDSPE on TLC as visualized with ninhydrin. This removal of excess DSPErequired at least three washings. The product, dansylated CPL, -tBoc(3), was dissolved in chloroform/methanol (2:1), washed with dilute HCland water, and precipitated using ether as described for (1). Yield: 575mg (96%). TLC (silica gel) chloroform/methanol (85:15) R_(f) 0.58. ¹HNMR (CDCl₃): δ 0.85 (t, 4H), 1.22 (s, 48H), 1.41 (s, 10H), 1.55 (t),2.27 (m[broad], 6H), 2.90 (m[broad], 6H), 3.04 (s, 8H), 3.27 (q), 3.61(s, 275H), 4.14 (m[broad]), 4.32 (d), 4.38 (d), 5.05 (s[broad]), 5.23(s[broad]), 5.58 (m[broad]), 7.37 (d, 1H), 7.49 (s[broad], 1H), 7.59 (t,2H), 8.24 (d, 1H), 8.50 (d, 1H), 8.59 (d, 1H) ppm.

[0288] Dansylated CPL₁ (4). Trifluoroacetic acid (TFA) (2 mL) was addedto a solution of dansylated CPL₁-tBoc (3) (550 mg, 121 μmmol) in 2 mL ofchloroform and stirred for 4 h at room temperature. The solution wasconcentrated to a thick paste and chloroform/ether washed three times.After the removal of ether, the solid was dissolved in 6 mL ofchloroform/methanol (2:1) and washed with 1.2 mL of 5% sodiumbicarbonate. The chloroform phase was extracted, dried and redissolvedin 6 mL chloroform/methanol (2:1) and washed with 1.2 mL distilledwater. The chloroform phase was concentrated to a thick paste and thepurified CPL₁ (4) was obtained through a chloroform/ether wash andvacuum dried. Yield: 535 mg (97%). TLC (silica)chloroform/methanol/water (65:25:4) R_(f) 0.76. ¹H NMR (CDCl₃). δ 0.85(t, 4H), 1.22 (s, 46H), 1.54 (m[broad], 8H), 2.23 (t, 6H), 2.84 (s, 9H),3.16 (m[broad], 3H), 3.26 (t, 3H), 3.61 (s, 263H), 3.98 (q), 4.17 (t),4.33 (d), 4.38 (d), 5.19 (s[broad]), 5.93 (d, 1H), 7.13 (d, 1H), 7.46(t, 1H), 7.52 (t, 1H), 8.15 (d, 1H), 8.43 (t, 2H) ppm.

[0289] Dansylated CPL₂-tBoc (5). A solution ofN_(α)N_(ε)-di-tBoc-L-lysine-N-hydroxysuccinimide ester (105 mg, 236μmol) in 2 mL dry chloroform was gradually added to a solution ofdansylated CPL₁ (4) (510 mg, 112 μmol) in 2 mL chloroform containing 200μL triethylamine and stirred at room temperature for 3 h. The completionof the reaction was indicated by the disappearance of primary amine asvisualized by ninhydrin assay on TLC. The reaction mixture wasconcentrated to a thick paste and chloroform/ether washed (˜3 times)until the disappearance of excessN_(α)N_(ε)-di-tBoc-L-lysine-N-hydroxysuccinimide ester as checked byTLC. The product was dissolved in 6 mL chloroform/methanol (2:1) andwashed with 1.2 mL 0.1 M HCl. The chloroform phase was extracted, dried,redissolved in 6 mL chloroform/methanol (2:1) and washed with 1.2 mLdistilled water. The chloroform phase was concentrated to a thick pasteand the purified compound was obtained through a chloroform/ether washand vacuum dried. Yield: 510 mg (96%). TLC (silica gel)chloroform/methanol (85:15) R_(f) 0.58. ¹H NMR (CDCl₃). δ 0.85 (t, 3H),1.22 (s, 44H), 1.41 (s, 20H), 1.56 (m[broad]), 1.78 (m[broad]), 2.27 (m,5H), 2.88 (s), 2.91 (s), 2.97 (s), 3.06 (s, 7H), 3.26 (t), 3.44 (t),3.62 (s, 252H), 3.97 (t), 4.05 (d), 4.13 (m), 4.33 (d), 4.38 (d), 4.68(s[broad]), 5.22 (s[broad]), 5.51 (s[broad]), 6.57 (t[broad], 1H), 7.39(d, 1H), 7.51 (s[broad], 1H), 7.60 (t, 2H), 8.26 (d, 1H), 8.53 (d, 1H),8.61 (d, 1H) ppm.

[0290] Dansylated CPL₂ (6). The synthesis of CPL₂ (6) was the same asthat of CPL₁ (4) by deprotecting dansylated CPL₂-tBoc (5) (490 mg, 103μmol). Yield: 478 mg (97%). TLC (silica) chloroform/methanol/water(65:25:4) R_(f) 0.63. ¹H NMR (CDCl₃). δ 0.85 (t, 3H), 1.22 (s, 42H),1.55 (m, 10H), 1.93 (s[broad], 4H), 2.24 (t, 5H), 2.85 (s, 8H), 3.26 (t,3H), 3.61 (s, 271H), 3.95 (q), 4.17 (s), 4.34 (s), 5.18 (s[broad], 1H),6.31 (d, 1H), 6.89 (s, 1H), 7.10 (d, 1H), 7.49 (m, 1H), 8.15 (d, 1H),8.34 (d, 1H), 8.47 (d, 2H) ppm.

[0291] Dansylated CPL₄-tBoc (7). The synthesis of CPL₄-tBoc (7) was thesame as that of CPL₂-tBoc (5) by reactingN_(α),N_(ε)-di-tBoc-L-lysine-N-hydroxysuccinimide ester (170 mg, 383μmol) with dansylated CPL₂ (6) (455 mg, 95 μmol). Yield: 475 mg (96%).TLC (silica gel) chloroform/methanol (85:15) R_(f) 0.58. ¹H NMR (CDCl₃).δ 0.85 (t, 3H), 1.22 (s, 43H), 1.40 (s, 39H), 1.71 (m[broad], 6H), 2.27(m, 5H), 2.88 (s), 2.90 (s), 2.95 (s), 3.05 (s, 10H), 3.25 (t, 3H), 3.43(s), 3.61 (s, 262H), 3.97 (t), 4.05 (d), 4.15 (m), 4.32 (d), 4.37 (d),4.51 (s[broad]), 4.75 (s[broad]), 4.90 (s[broad]), 5.23 (t[broad], 1H),5.52 (s[broad]), 5.80 (s[broad], 1H), 7.15 (m[broad], 1H), 7.38 (d, 1H),7.50 (s, 1H), 7.59 (t, 2H), 8.25 (d, 1H), 8.51 (d, 1H), 8.60 (d, 1H)ppm.

[0292] Dansylated CPL₄ (8). The synthesis of CPL₄ (8) was the same asthat of CPL₁ (4) by deprotecting dansylated CPL₄-tBoc (7) (450 mg, 86μmol). Yield: 440 mg (97%). TLC (silica) chloroform/methanol/water(65:25:4) R_(f) 0.19. ¹H NMR (CDCl₃). δ 0.85 (t), 1.22 (s), 1.53(m[broad]), 2.34 (m[broad]), 2.86 (s), 3.26 (t), 3.62 (s), 3.87(s[broad]), 3.97 (t), 4.17 (s[broad]), 4.33 (d), 5.18 (s[broad]), 7.15(d), 7.43 (s), 7.51 (t), 8.15 (d), 8.32 (d), 8.48 (d), 9.05 (s[broad])ppm.

[0293] Dansylated CPL₈-tBoc (9). The synthesis of CPL₈-tBoc (9) was thesame as that of CPL₂-tBoc (5) by reactingN_(α)N_(ε)-di-tBoc-L-lysine-N-hydroxysuccinimide ester (70 mg, 158 μmol)with dansylated CPL₄ (8) (100 mg, 19 μmol). Yield: 112 mg (96%). TLC(silica gel) chloroform/methanol (85:15) R_(f) 0.58. ¹H NMR (CDCl₃). δ0.84 (t, 3H), 1.08 (s), 1.21 (s, 39H), 1.39 (s, 75H), 1.66 (m [broad]),2.26 (m, 4H), 2.89 (s, 4H), 3.06 (s, 11H), 3.25 (t, 3H), 3.43 (s), 3.49(s), 3.60 (s, 248H), 3.96 (t), 4.04 (d), 4.12 (t), 4.31 (d), 4.36 (m),5.19 (m [broad]), 6.77 (m [broad], 1H), 6.91 (s [broad], 1H), 7.24(CHCl₃), 7.41 (d), 7.50 (s [broad]), 7.60 (t), 8.25 (d, 1H), 8.53 (d,1H), 8.63 (d, 1H) ppm.

[0294] Dansylated CPL₈ (10). The synthesis of CPL₈ (8) was the same asthat of CPL₁ (4) by deprotecting dansylated CPL₈-tBoc (9) (50 mg, 8μmol). Yield: 48 mg (96%). TLC (silica) chloroform/methanol/water(65:25:4) R_(f) 0.13. ¹H NMR (CDCl₃). δ0.85 (t, 3H), 1.22 (s, 34H), 1.52(s [broad]), 2.23 (s [broad]), 2.86 (d), 3.27 (d), 3.61 (s, 274H), 3.96(t), 4.18 (m [broad]), 7.14 (s [broad]), 7.24 (CHCl₃), 7.50 (m [broad]),8.12-8.27 (s [broad]), 8.47 (m [broad]) ppm.

[0295] C. Results and Discussion

[0296] The CPL were synthesized by repeated coupling reaction stepsinvolving amines and NHS-activated carbonate groups as outlined in FIG.29. This consists of (a) incorporating the dansyl fluorescent label tothe hydrophilic PEG spacer, (b) coupling of the DSPE anchor, and (c)attachment of the cationic headgroup to the lipid. Theheterobifunctional PEG polymer tBoc-NH-PEG₃₄₀₀-CO₂—NHS (MW 3400), waschosen for two reasons. Firstly, it was commercially available.Secondly, it is insoluble in ether that provided a very convenient meansof purifying its derivatives, 1-10. Other reagents were used in excessto ensure the complete conversion of the PEG polymer to its derivatives.The excess reagents were soluble in ether and therefore could be removedby washing in ether during purification.

[0297] Incorporation of the fluorescent label, N_(ε)-dansyl lysine, tothe PEG polymer by coupling the α-amino group of dansyl lysine with theNHS activated carbonate of PEG gave the lysine derivative 1. The DSPEanchor was coupled via intermediate 2 that was formed by theesterification of 1 using NHS and DCC. The resulting PEG lipid, 3, wasdeprotected by removing the tBoc to form CPL₁, 4, with one positivecharge. The positive charges in the other CPL are carried by the aminogroups of lysine. Here, the NHS activated and di-tBoc protected lysinewas attached to the free amino function of CPL₁ to form intermediate 5which, upon deprotection, yielded CPL₂, 6, with two positive charges.The attachment of two lysine residues to the amino groups of CPL₂ viaintermediate 7 gave CPL₄, 8, with four positive charges. Thus, CPL₈, 10,with eight positive charges was synthesized with the attachment of fourlysine residues as the headgroup. As can be seen, this provides a veryconvenient means of synthesizing multivalent CPL that are of particularinterest for non-viral drug delivery applications.

[0298] The structures of the purified intermediates and CPL in FIG. 29were verified by ¹H NMR spectroscopy and chemical analysis. The ¹H NMRspectra showed well-resolved resonances for the PEG, tBoc and acylchains of DSPE at approximately 3.61, 1.41 and 1.21 ppm, respectively,and for the resonances of the dansyl moiety (aromatic protons at 7.1-8.5ppm; methyl protons at 2.8-3.0 ppm). From the integrated signalintensities of the former three peaks, it was found that the ratio oftBoc/PEG or tBoc/DSPE was 1.0, 2.1, 4.0, and 8.1 for CPL₁-tBoc,CPL₂-tBoc, CPL₄-tBoc, and CPL₈-tBoc, respectively. As each tBoc isattached to an amino group, this gives the number of amino groups in theheadgroup of each CPL relative to the CPL₁. That essentially identicalresults were obtained using the ratios of tBoc relative to both PEG andDSPE demonstrates the presence of lipid and polymer in correctproportion to the headgroup. The complete cleavage of the tBocprotecting groups was verified by the loss of tBoc NMR peaks andchemical analysis which determined the ratio of primary amine tophosphate in each of the CPL by using the fluorescamine and phosphorusassays. The amine/phosphate ratios for CPL₁, CPL₂, CPL₄, and CPL₈ werefound to be 1.0, 2.2, 3.7, and 8.0, respectively. These correspondedwell with the expected number of positive charge bearing amino groups ofthe respective CPL.

[0299] The CPL described here possess several attributes which mayincrease their usefulness relative to other cationic lipids. Firstly,the phospholipid anchor will readily allow efficient incorporation ofCPL into liposomal systems. Secondly, the dansyl label will permitaccurate and convenient quantification of the CPL in the bilayer usingfluorescence techniques. Finally, the valency of the cationic headgroupin the CPL can easily be modified using lysine residues.

VIII. Example VIII

[0300] A. General Overview

[0301] The synthesis of a fluorescent cationic poly(ethylene glycol) (MW1000) lipid conjugates (CPL)¹ is described. The procedure is verysimilar to that of PEG 3400 described in detail previously. However thelower molecular weight PEG derivatives may not be insoluble in ether,and therefore could not be readily purified by ether wash as before. Thesynthetic procedure is similar to the one outlined in FIG. 29.

[0302] B. Abbreviations

[0303] tBoc, tert-butyloxycarbonyl; tBoc-NH-PEG₁₀₀₀-CO₂—NHS, tBocprotected and NHS activated PEG₁₀₀₀; CPL, cationic poly(ethylene glycol)lipid conjugate; CPL₁, CPL with one positive charge; CPL₂, CPL with twopositive charges; CPL₄, CPL with four positive charges; DCC,N,N′-dicyclohexyl-carbodiimide; DCU, dicyclohexyl urea; NHS,N-hydroxysuccinimide; di-tBoc-lysine-NHS,N_(α),N_(ε)-di-tBoc-L-lysine-N-hydroxysuccinimide ester; DSPE,1,2-distearoyl-sn-glycero-3-phosphoethanolamine; PEG₁₀₀₀, poly(ethyleneglycol) with an average MW of 1000; TFA, trifluoroacetic acid.

[0304] C. Materials and Reagents

[0305] tBoc-NH-PEG₁₀₀₀-CO₂—NHS was obtained from Shearwater Polymers(Huntsville, Ala.). N_(α),N_(ε)-di-tBoc-L-lysine-N-hydroxysuccinimideester, N_(ε)-dansyl-L-lysine, N-hydroxysuccinimide (NHS), andN,N′-dicyclohexyl-carbodiimide (DCC) were purchased from Sigma-AldrichCanada (Oakville, ON). 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine(DSPE) was obtained from Northern Lipids (Vancouver, BC). Fluorescaminewas obtained from Molecular Probes (Eugene, Oreg.). Trifluoroaceticacid, diethyl ether, methanol, triethylamine, and chloroform wereobtained from Fisher Scientific (Vancouver, BC). All other reagents wereused without further purification.

[0306] tBoc-NH-PEG₁₀₀₀-CO₂—(N_(ε)-dansyl)lysine (1).tBoc-NH-PEG₁₀₀₀-CO₂—NHS (500 mg, 500 μmol) in 3 mL of dry chloroform wasadded slowly to a solution of N_(ε)-dansyl-L-lysine (200 mg, 536 μmol)in 1.5 mL of methanol and 300 μL of triethylamine. After the reactionmixture was stirred at room temperature for 3 h, the solvent was removedunder a N₂ stream and further dried under vacuum. The crude product wasdissolved in 6 mL of chloroform/methanol (2:1 v/v), washed once with 1.2mL of 0.5 M HCl and twice with 1.2 mL of distilled water. The chloroformphase was extracted, dried to a thick paste andtBoc-NH-PEG-CO₂—(N_(ε)-dansyl)lysine (I) was obtained as a light yellowsolid. Yield: 600 mg (95%). TLC (silica gel) chloroform/methanol (85:15v/v): R_(f) 0.50.

[0307] Dansylated CPL₁-tBoc (3). First,tBoc-NH-PEG₁₀₀₀-CO₂—(N_(ε)-dansyl)lysine-NHS (2) was prepared asfollows. A solution of tBoc-NH-PEG₁₀₀₀-CO₂—(N_(ε)-dansyl)lysine (1) (600mg, 474 μmol) and NHS (113 mg, 982 mmol) in 2 mL of dry chloroform wasadded to DCC (150 mg, 728 μmol) dissolved in 1 mL of dry chloroform. Thereaction mixture was stirred for 5 h at room temperature. Theby-product, dicyclohexyl urea (DCU), was filtered using a Pasteurpipette with a cotton plug. The filtrate, containingtBoc-NH-PEG₁₀₀₀-CO₂—(N_(ε)-dansyl)lysine-NHS (2), was slowly added to asolution of DSPE (365 mg, 488 μmol) in 3 mL of dry chloroform and 300 μLof triethylamine. The dissolution of DSPE in dry chloroform andtriethylamine required warming to 65° C. After the reaction mixture wasstirred overnight at room temperature, it was filtered to remove someprecipitate (unreacted DSPE) and dried to a viscous paste. The paste wasdissolved in chloroform/methanol (2:1), washed with dilute HCl and wateras before. The product, dansylated CPL₁-tBoc (3), was obtained after theremoval of solvent and precipitated using 10 mL of ether. Yield: 900 mg(96%). TLC (silica gel) chloroform/methanol (85:15) R_(f) 0.58.

[0308] Dansylated CPL₁ (4). Trifluoroacetic acid (TFA), 3 mL, was addedto a solution of dansylated CPL₁-tBoc (3) (900 mg, 456 μmol) in 3 mL ofchloroform and stirred for 4 h at room temperature. The solution wasconcentrated to a thick paste and chloroform/ether washed three times.After the removal of ether, the solid was dissolved in 6 mL ofchloroform/methanol (2:1) and washed twice with 1.2 mL of 5% sodiumbicarbonate and twice with 1.2 mL distilled water. The chloroform phasewas concentrated to a thick paste and the purified CPL₁ (4) was obtainedthrough a chloroform/ether wash and vacuum dried. Yield: 750 mg (88%).TLC (silica) chloroform/methanol/water (65:25:4) R_(f) 0.72.

[0309] Dansylated CPL₂-tBoc (5). A solution ofN_(α)N_(ε)-di-tBoc-L-lysine-N-hydroxysuccinimide ester (350 mg, 789[mol) in 3 mL dry chloroform was gradually added to a solution ofdansylated CPL₁ (4) (750 mg, 400 μmol) in 3 mL chloroform containing 300μL triethylamine and stirred at room temperature for 3 h. The completionof the reaction was indicated by the disappearance of primary amine asvisualized by ninhydrin assay on TLC. The reaction mixture wasconcentrated to a thick paste, redissolved in 6 mL chloroform/methanol(2:1) and washed once with 1.2 mL 0.5 M HCl four times with 1.2 mLdistilled water. The chloroform phase was extracted and dried. Nofurther purification was performed. Yield: 700 mg (81%). TLC (silicagel) chloroform/methanol (85:15) R_(f) 0.58.

[0310] Dansylated CPL₂ (6). The synthesis of CPL₂ (6) was the same asthat of CPL₁ (4) by deprotecting dansylated CPL₂-tBoc (5) (700 mg, 318μmol). Yield: 650 mg (92%). TLC (silica) chloroform/methanol/water(65:25:4) R_(f) 0.63.

[0311] Dansylated CPL₄-tBoc (7). The synthesis of CPL₄-tBoc (7) was thesame as that of CPL₂-tBoc (5) by reactingN_(α),N_(ε)-di-tBoc-L-lysine-N-hydroxysuccinimide ester (500 mg, 1127μmol) with dansylated CPL₂ (6) (650 mg, 292 μmol). Besides washing withdilute HCL and water no further attempts were made to purify CPL₄-tBocbefore deblocking to generate CPL₄. Yield: 800 mg (Crude). TLC (silicagel) chloroform/methanol (85:15) R_(f) 0.58 (dansyl peak only).

[0312] Dansylated CPL₄ (8). The synthesis of CPL₄ (8) was the same asthat of CPL₁ (4) by deprotecting dansylated CPL₄-tBoc (7) (800 mg). Thefinal product was purified by column chromatography using silica gel 60,70-230 mesh, and chloroform/methanol/ammonia solution (65:25:4 v/v).Yield: 300 mg (38%). TLC (silica) chloroform/methanol/water (65:25:4)R_(f) 0.15.

IX. Example IX

[0313] A. General Overview

[0314] We show here that CPL₄ can be inserted into preformed SPLP andthat the resulting SPLP-CPL₄ exhibit improved uptake and markedlyimproved in vitro transfection potency in BHK cells. These resultsestablish that the SPLP system is intrinsically a highly potenttransfection vector.

[0315] B. Materials and Methods

[0316] 1. Preparation of SPLP, SPLP-CPL₄, and Complexes

[0317] (i). SPLP: SPLP composed of DOPE:DODAC:PEG-CerC₂₀ (84:6:10) andcontaining the plasmid pLuc, a modified marker gene expressingluciferase, was supplied by INEX Pharmaceuticals Inc.

[0318] (ii) SPLP-CPL₄: Dansylated CPL₄ was prepared in our laboratoryand incorporated into SPLP as follows: SPLP at a dose of 500 nmol lipidwas incubated with different amounts of CPL₄ (12.5, 19, and 30 nmol) at60° C. for 2 to 3 hours in Hepes Buffered Saline, pH 7.5 (HBS) toachieve a final incorporation of 2, 3, and 4 mol %, respectively.SPLP-CPL was separated from unincorporated CPL by gel filtrationchromatography on a Sepharose CL-4B column equilibrated in HBS.Fractions (1 mL) were collected and assayed for CPL, phospholipid andDNA contents. Fractions containing all three components were pooled andconcentrated for use in transfection and uptake studies. The samplesfrom the column were greatly aggregated. To deaggregate the systems,addition of CaCl₂ or MgCl₂ was required. Experiments to determine theoptimal amount of cation for deaggregation will be described later inthe Methods.

[0319] CPL Assay: The presence of CPL was determined by measuring thefluorescence of the dansyl group in CPL on a Perkin Elmer LS52Luminescence spectrophotometer using λ_(ex)=340 nm and λ_(em)=510 nmwith excitation and emission slit widths of 10 and 20 nm, respectively.Fluorescence of the dansyl was quantified using a standard curve ofdansylated CPL in HBS.

[0320] Phospholipid Assay: Phospholipid was determined by firstextracting the lipids from SPLP using the Bligh-Dyer technique. and thenmeasuring phosphate in the organic phase according to the Fiske-Subbarowmethod (see, Bligh E G, Dyer W J A rapid method of total lipidextraction and purification. Can J Biochem Physiol 1959; 37: 911-917;and Fiske C H, Subbarow Y. The calorimetric determination ofphosphorous. J Biol Chem 1925; 66: 375-400.).

[0321] DNA Assay: DNA content was measured using the PicoGreen Assay kit(Molecular Probes, Eugene, Oreg.) as previously described. (see, MokKWC, Lam AMI, Cullis PR. Stabilized plasmid-lipid particles: factorsinfluencing plasmid entrapment and transfection properties. BiochimBiophys Acta 1999; 1419: 137-150).

[0322] (iii) Complexes: The complexes were prepared, at a charge ratioof 1.5:1+ve/−ve, by mixing 25 μL of DOPE:DODAC (0.8 mM), kindly suppliedby Inex, with 25 μL of 88 μg/mL pLuc, also supplied by Inex, followed byincubation for 30 min before addition to cells.

[0323] 2. Preparation of SPLP Containing 0.5 mol % of Rh-PE for OptimalInsertion Time Determination and Lipid Uptake Experiments

[0324] SPLP were prepared as described by Wheeler et al. (see, Wheeleret al., Gene Therapy; 6:271-281 (1999)) with a few modifications. Thelipids DOPE, PEG-CerC₂₀, DODAC, and rhodamine-DOPE (Rh-PE), all stocksin CHCl₃, were mixed together in a molar ratio of (83.5:10:6:0.5) andthe CHCl₃ was completely evaporated. The resulting lipid film wasdissolved in 20 mM octyl glucopyranoside (OGP) and 200 μg/mL of plasmidDNA was added to a total volume of 1 mL. The OGP was dialysed from thesample in a dialysis bag with two changes of buffer (HBS) over 48 hours.The resulting sample was passed down a DEAE Sepharose column and theeffluent was run on a discontinuous sucrose gradient as describedpreviously. (see, Gabizon A, Papahadjopoulos D. Liposome formulationswith prolonged circulation time in blood and enhanced uptake by tumors.Proc Natl Acad Sci USA 1988; 85: 6949-6953.). The resultingrhodamine-labeled SPLP possessed a DNA/Lipid ratio of ˜60 μg/μmol.

[0325] 3. Determination of Optimal Incubation Time for Insertion of CPL4into SPLP

[0326] To determine the time required for optimal insertion of CPL₄ intoSPLP, 5 mol % of CPL₄ (0.3 nmol) was mixed with 6 nmol of SPLP(containing 0.5 mol % Rh-PE) in a total volume of 1.5 mL and incubatedin a 60° C. water bath. At time points (30 min, 1 h, 2 h, 3 h, and 4 h),250 μL of the mixture was run down a Sepharose CL-4B column equilibratedwith HBS. The fractions possessing fluorescent dansyl were combined andthe dansyl fluorescence was measured using the parameters describedabove while the rhodamine fluorescence was measured using λ_(ex)=560 nm,λem=590 nm, and excitation and emission slit widths of 10 and 20 nm,respectively. These measurements were also made on a small fraction ofthe original solution before the column. The dansyl/rhodamine ratios arecalculated for both the initial and final samples to determine thepercentage of the initial 5 mol % that was inserted.

[0327] 4. Deaggregation of SPLP-CPL4 Using CaCl2 and MgCl2

[0328] As stated above, the preparation of SPLP-CPL₄ results inaggregation of the particles. To deaggregate the system an increase inionic strength is required. This was achieved by the addition ofincreasing amounts of CaCl₂ or MgCl₂ (500 mM stock solution) to asolution of SPLP-CPL₄. To 60 μL of SPLP-CPL₄ (3 mM lipid) was added 360μL of HBS in a Nicomp tube. The mean diameter±standard deviation of theSPLP-CPL₄ (0 mM Cation) was then determined by QELS using a Nicomp Model270 Submicron Particle Sizer. Then the salt (CaCl₂ or MgCl₂) was addedto concentrations from 20 mM to 70 mM. At each interval the meandiameter±standard deviation was determined by QELS. The mean diameter ofthe particles hardly changes with increasing [Cation], however, the QELSGaussian distribution gets broader. Therefore, the standard deviationswere used as a measure of deaggregation.

[0329] 5. Size Determination of SPLP-CPL4 and SPLP

[0330] Freeze-fracture EM was performed on the SPLP-CPL₄ (no CaCl₂),SPLP-CPL₄+40 mM CaCl₂, and SPLP, according to Wheeler et al. (see,Wheeler J J et al. Stabilized plasmid-lipid particles: construction andcharacterization. Gene Therapy 1999; 6: 271-281.). The SPLP-CPL₄contained 4 mol % CPL₄. The micrographs of SPLP-CPL₄, in the presenceand absence of CaCl₂, were compared to show the visual effect of Ca²⁺ onthe aggregation. Vesicle diameters of the SPLP-CPL₄+40 mM CaCl₂ and SPLPwere analyzed by QELS using a Nicomp Model 270 Submicron Particle Sizer.

[0331] 6. Serum Stability of SPLP-CPL4 Particles

[0332] The serum stability of the SPLP-CPL containing various % of CPLwere determined by mixing the particles with mouse serum to a finalserum concentration of 50% v. These mixtures were then incubated for 0,1, 2, or 4 hours at 37° C. At these time points, a volume of the mixturecontaining about 1 μg of plasmid DNA was removed and the DNA wasextracted from the lipid and protein using a phenol:chloroformextraction. The resulting DNA solutions were then run on a 1% agarosegel following which the DNA was transferred to nitrocellulose and aSouthern blot was performed.

[0333] 7. Lipid Analysis of SPLP-CPL₄

[0334] To determine the loss of PEG-CerC₂₀ from the SPLP during theinsertion of CPL₄, lipid was extracted for the SPLP sample and SPLP-CPL₄sample by the Bligh-Dyer extraction. The mixtures were then passedthrough an HPLC and were assayed for DOPE and PEG-CerC₂₀ by NorthernLipids, Inc (Vancouver, BC). The DOPE:PEG-CerC₂₀ ratios for the SPLP-CPLwas compared to that for the SPLP and the amount of PEGylated lipid inthe outer monolayer of the SPLP was determined.

[0335] 8. Uptake Studies

[0336] For all in vitro experiments, the cells used were a transformedBHK cell line (tk-). For the uptake studies, 1×10⁵ BHK cells were grownon 12-well plates overnight in 2 mL of complete media (DMEM+10% FBS) at37° C. in 5% CO₂. SPLP, SPLP-CPL₄+40 mM CaCl₂, or DOPE:DODAC complexes(200 μL), each containing 0.5 mol % Rh-PE as lipid marker were mixedwith 800 μL of complete media and this mixture was added to the top ofthe cells at a lipid dose of 20 μM. After incubation at 37° C. for 2, 4,6, or 8 hours, the cells were washed with PBS and lysed with 600 μL oflysis buffer (0.1% Triton X-100 in PBS). The rhodamine fluorescence ofthe lysate was measured in a 1.0 mL microcuvette on a Perkin-Elmer LS52Luminescence Spectrophotometer using a λ_(ex) of 560 nm and a λ_(em) of600 nm with slit widths of 10 and 20 nm, respectively. An emissionfilter of 430 nm was also used. Lipid uptake was determined bycomparison of the fluorescence in the lysate to that of a lipid standardand normalized to the amount of cells as determined by the BCA proteinassay (Pierce, Rockford, Ill.). Where indicated, fluorescencemicrographs were taken on an Axiovert 100 Zeiss Fluorescent microscope(Carl Zeiss Jena GmbH) using a rhodamine filter from Omega Opticals(Brattleboro, Vt.) with the following specifications, λ_(ex)=560±20 nm,600 nm LP, and DC 590 nm.

[0337] 9. Effect of Type and Concentration of Cation on Lipid Bindingand Uptake

[0338] This uptake experiment was performed with the same SPLP-CPL₄(containing 0.5 mol % Rh-PE) as above. 5×10⁴ BHK cells were platedovernight in 1 mL of complete media in 24-well plates. The SPLP-CPL₄ (40nmol) was mixed with CaCl₂ or MgCl₂ at various initial concentrations of20 mM to 70 mM in a total volume of 100 μL. To this was added 400 μL ofcomplete media resulting in final [Cation] of 4 mM to 14 mM. Thismixture was then added to the top of the cells and the cells incubatedfor 4 hours. After incubation the cells were washed twice with PBS and600 μL of lysis buffer (0.1% Triton X-100 in PBS) was added. As above,the rhodamine fluorescence was measure and the lipid uptake wasdetermined comparing the resulting fluorescence to that of a standardsample containing a known amount of lipid. The resulting values werethen normalized to the number of cells by measuring the protein contentusing the BCA protein assay kit.

[0339] 10. Transfection Studies

[0340] 1×10⁴ BHK cells were plated in 96-well plates in 150 μL completemedia and incubated overnight at 37° C. in 5% CO₂. SPLP and SPLP-CPL,containing between 2 and 4 mol % CPL, were prepared to deliver 0.5 μg ofDNA in a total volume of 20 μL using HBS (SPLP), or HBS+40 mM CaCl₂(SPLP-CPL₄) and were added to 90 μL of complete media. Samples wereincubated with the cells for 4 hours. The transfection media was thenreplaced with complete media for a complete 24 hour incubation. Cellswere then lysed with 100 μL of lysis buffer, and 40 μL of the lysate wastransferred to a 96-well luminescence plate. Luciferase activity wasdetermined using a Lucifcrase reaction kit (Promega, Madison, Wis.), aluciferase standard (Boehringer-Manheim), and a ML3200 microtiter plateluminometer from Molecular Dynamics (Chantilly, Va.). Activity wasnormalized to the number of cells as measured by the BCA protein assay(Pierce, Rockford, Ill.). From the uptake and transfection experimentsabove, it was determined that 4 mol % CPL₄ in SPLP-CPL₄ gave optimalresults. Thus, the rest of the experiments were performed with SPLP-CPL₄containing 4 mol % CPL₄.

[0341] 11. Time Course for the transfection of SPLP-CPL versus SPLP andComplexes

[0342] Samples and cells were prepared as described for the abovetransfection study, and incubated together at 37° C. As well, Lipofectin(Gibco BRL, ) complexes containing pLuc were prepared at a charge ratioof 1.5:1. At 4, 9, and 24 hours, the transfection media was removed andin the case of the 4 and 9 hour transfections, replaced with completemedia for a complete 24-hour incubation. At 24 h, all cells were lysedand assayed for luciferase activity and protein content (BCA assay), asabove.

[0343] 12. Transfection Potency and Toxicity of SPLP-CPL4

[0344] BHK cells were incubated with SPLP, SPLP-CPL₄+40 mM CaCl₂, andLipofectin complexes for 24 or 48 hours. After the incubation period thecells were immediately lysed and the luciferase activity was measuredand was normalized to the amount of protein present, as above.

[0345] As a rough measure of cell survival at the above time points, theprotein concentration after cell lysis at 24 and 48 hours was measuredand compared for the SPLP-CPL₄+40 mM CaCl₂ and the Lipofectin complexes.

[0346] 13. Comparison of Effect of Ca2+ and Mg2+ on Transfection of BHKCells

[0347] Cells were plated and used as above. SPLP-CPL₄ (5.0 μg/mL) witheither CaCl₂ or MgCl₂ at concentrations of 20 mM to 70 mM were combinedin a volume of 20 μL and mixed with complete media, resulting in final[Cation] of 4 mM to 14 mM. Following incubation on the cells for 48hours, the cells were washed and lysed, and the luciferase activity andprotein content were measured as above.

[0348] 14. Measurement of Transfection Efficiency of SPLP-CPL4

[0349] The transfection efficiency of the SPLP-CPL was measured bypreparing SPLP-CPL₄ containing encapsulated pEGFP (kindly supplied byInex), that expresses GFP (green fluorescence protein), using thedetergent dialysis procedure. (see, Wheeler et al. supra). 400 μg/mL ofpEGFP was encapsulated within 10 mM DOPE:PEG-CerC20:DODAC (84:10:6),followed by the insertion of 4 mol % of CPL₄. DOPE:DODAC complexes andLipofectin complexes containing pEGFP were also prepared at a chargeratio of 1.5:1. The transfections were performed as described earlier ata DNA dose of 5.0 μg/mL. Following incubation of the samples for 24 and48 hours, the transfection media was removed, the cells were washed, andfresh media was added to the cells. The cells were then viewed under theZeiss fluorescence microscope. The total number of cells within theframe were counted; then the number of cells expressing the GFP werecounted using a fluorescein filter (Omega Opticals) with the followingspecifications, λ_(ex)=470±20 nm, λ_(em)=535±22.5 nm, and DC ˜500 nm.The efficiency of transfection is the number of cells expressing the GFPdivided by the total number of cells.

[0350] C. Results and Discussion

[0351] 1. SPLP-CPL4 Aggregate Following Insertion of CPL4 andDe-aggregate Following Addition of Divalent Cations.

[0352] LUV containing CPL tend to aggregate, and that this aggregationcan be inhibited by increasing the ionic strength of the medium. It wasfound that SPLP-CPL₄ were also susceptible to aggregation, and that thisaggregation could be reversed by adding NaCl, CaCl₂ or MgCl₂ to theSPLP-CPL₄ formulation. This effect is illustrated in FIG. 31 which showsthe effect of the addition of CaCl₂ and MgCl₂ on aggregation ofSPLP-CPL₄ as monitored by the change in the standard deviation of themean diameter of the particles measured by quasi-elastic lightscattering (QELS). For both cations the standard deviation decreaseswith increasing cation concentration with optimal de-aggregationoccurring above 30 to 40 mM. This behavior could also be visualized byfreeze-fracture electron microscopy. Freeze-fracture micrographs of SPLPreveal small monodisperse particles, whereas SPLP-CPL₄ prepared in theabsence of CaCl₂ are highly aggregated. The addition of 40 mM CaCl₂reverses this aggregation to produce monodisperse particles similar tothe SPLP preparation. For details of sample preparation and electronmicroscopy, (see, Wheeler et al., Gene Therapy; 6:271-281 (1999)).

[0353] The sizes of SPLP and SPLP-CPL₄ in the presence of CaCl₂ werecompared using QELS and freeze-fracture electron microscopy. QELSstudies revealed the mean diameter of SPLP and SPLP-CPL₄ to be 80±19 nmand 76±15 nm, respectively, whereas the freeze-fracture studiesindicated to diameters of 68±11 nm and 64±14 nm. These values for SPLPare in close agreement with previous studies.

[0354] 2. Chemical Composition and Stability of SPLP-CPL4.

[0355] The lipid composition of SPLP-CPL₄ and SPLP are given in Table 9below: TABLE 9 Loss of PEG-CerC₂₀ from SPLP following CPL₄ insertion.[DOPE] [PEG-CerC₂₀] % PEG-CerC₂₀ after (mM) (mM) DOPE:PEG-C₂₀ insertion0.786 0.0714 ± 0.0004 11.0 ± 0.1 79.7 ± 0.9% (81.6:7.4; mol) (x = 5.9 ±0.1 mol %) SPLP-CPL₄ 0.790 ± 0.007 0.0572 ± 0.0003 13.8 ± 0.1 (81.6:x;mol)

[0356] By analysis of the SPLP itself, the molar ratio ofDOPE:PEG-CerC₂₀ was 11.0(±0.1):1. This corresponds to a system ofDOPE:PEG-CerC₂₀:DODAC of (81.6:10.9:7.4). From the results, 79.7±0.9% ofthe PEG-CerC₂₀ remains following CPL₄ insertion. This corresponds to afinal mol % of PEG-CerC₂₀ of 5.9±0.1 mol %. This means that about1.5±0.1 mol % of PEG-CerC₂₀ was replaced during the insertion of CPL₄.If we assume that on the inner leaflet and outer leaflet the same amountof PEG-CerC₂₀ is initially present at 7.4 mol %, the outer leaflet willpossess 4.4±0.1 mol % of PEG-CerC₂₀ after insertion. Since we inserted˜4.5 mol % CPL₄ into SPLP (9.0 mol % in the outer leaflet), resulting ina total of 13.4±0.1 mol % of total PEG in the outer leaflet.

[0357] The stability of SPLP and SPLP-CPL₄ in 50% mouse serum for up to4 hours. In all cases, the DNA was completely protected from serumdegradation.

[0358] 3. SPLP-CPL₄ Exhibit Enhanced Uptake into BHK Cells andDramatically Enhanced Transfection Potency.

[0359] The next set of experiments was aimed at determining theinfluence of incorporated CPL₄ on the uptake of SPLP into BHK cells andthe resulting transfection potency of the SPLP-CPL₄ system. SPLPcontaining up to 4 mol % CPL₄ were prepared in the presence of 40 mMCaCl₂ and were added to BHK cells (final CaCl₂ concentration 8 mM) andincubated for varying times. The cells were then assayed for associatedSPLP-CPL₄ as indicated in Methods. As shown in FIG. 32, uptake of SPLPthat contain no CPL₄ is minimal even after 8 h of incubation, howeveruptake is dramatically improved for SPLP containing 3 mol % or higherlevels of CPL₄. For example, SPLP containing 4 mol % CPL₄ exhibitaccumulation levels at 8 h that are approximately 50-fold higher thanachieved for SPLP. This enhanced uptake can be visually detected usingfluorescence micrographs of BHK cells following incubation withrhodamine-labeled SPLP and SPLP-CPL₄ for 4 h. The presence of 4 mol %CPL₄ clearly results in improved levels of cell-associated SPLP.

[0360] The transfection properties of SPLP, SPLP-CPL₄ and plasmidDNA-cationic lipid complexes (DODAC/DOPE; 1:1; 1.5:1+ve/−ve c.r.) wereexamined using the incubation protocol usually employed for complexes.This consisted of incubation of 10⁴ BHK cells with SPLP, SPLP-CPL₄ andcomplexes containing 0.5 μg pCMVLuc for 4 h, followed by removal ofSPLP, SPLP-CPL₄ or complexes that are not associated with the cells,replacement of the media, incubation for a further 20 h and thenassaying for luciferase activity. The SPLP-CPL₄ preparations contained 7mM CaCl₂ in the incubation medium. As shown in FIG. 33, the presence ofthe CPL₄ resulted in dramatic increases in the transfection potencies ofthe SPLP system. SPLP-CPL₄ containing 4 mol % CPL₄ exhibited luciferaseexpression levels some 3×10³ higher than achieved with SPLP. (see, Moket al., Biochim Biophys Acta, 1419:137-150 (1999)).

[0361] 4. Ca2+ is Required for Transfection Activity of SPLP-CPL₄.

[0362] It was of interest to determine the influence of Ca²⁺ on thetransfection activity of SPLP-CPL₄. SPLP containing 4 mol % CPL₄ wereincubated with BHK cells for 48 h in the presence of 0-14 mM MgCl₂ andCaCl₂ and the luciferase activities then determined. As shown in FIG.34, the transfection activity was influenced by the presence of Ca²⁺ inthe transfection medium. At the optimum CaCl₂ concentration of 10 mM,SPLP-CPL₄ exhibited transfection potencies that were more than 10⁴ timeshigher than if MgCl₂ was present.

[0363] Uptake of SPLP-CPL₄ into BHK cells was monitored following a 4 hincubation in the presence of 0-14 mM MgCl₂ and CaCl₂. As shown in FIG.35 the amount of SPLP-CPL₄ taken up by BHK cells is the same for bothMg²⁺ and Ca²⁺-containing media. The uptake of the SPLP-CPL₄ decreases asthe concentration of divalent cations increases, which likely arises dueto shielding of the negatively charged binding sites for the CPL₄ on thesurface of the BHK cells.

[0364] 5. SPLP-CPL₄ Exhibit Transfection Potencies in Vitro that areComparable to or Greater than Achieved Using Complexes.

[0365] The results shown in FIG. 33 indicating that complexes give riseto ˜100-fold higher levels of transfection than SPLP-CPL₄ were obtainedfor a fixed 4 h incubation time with the BHK cells, followed by a 20 hhold time to achieve maximum expression. Given that the SPLP-CPL₄ arestable systems it is likely that uptake into the BHK cells wouldcontinue over extended time periods. The transfection levels achievedwhen the incubation time of the SPLP-CPL₄ and the complexes with the BHKcells was extended to 8 and 24 h, followed by hold times of 16 and 0 hrespectively were examined. Two types of plasmid DNA-cationic lipidcomplexes were used, namely DOPE:DODAC (1:1) complexes (1.5:1, c.r.) andcomplexes obtained using the commercial transfection reagent Lipofectin(DOPE/DOTMA [1:1] complexes, 1.5:1 c.r.). As shown in FIG. 36, thetransfection potency of the SPLP-CPL₄ increases markedly with increasedincubation times, suggesting that a limiting factor for transfectionachieved at a 4 h incubation time was the rate of uptake of theSPLP-CPL₄ system. At the 24 h incubation time transfection levels areachieved that are comparable to those achieved by Lipofectin orDOPE/DODAC complexes.

[0366] Further experiments were conducted to determine transfectionlevels after 24 and 48 h incubation times with luciferase activitiesassayed immediately following the incubation period. As shown in FIG.37A the activity of Lipofectin (DOPE/DOTMA; 1:1) complexes leveled offat ˜2000 ng/mg after 24 h. In contrast, the activity of SPLP-CPL₄formulation continued to increase as the incubation time was increased,achieving luciferase expression levels corresponding to 4000 ng/mg at 48h. This activity is approximately 10⁶ times higher than observed forSPLP (in the absence of Ca²⁺) and almost double the levels that can beachieved by Lipofectin complexes. Similar results were obtained for theDOPE:DODAC complexes.

[0367] 6. SPLP-CPL4 are Non-Toxic and Efficient Transfection Agents.

[0368] It is well known that plasmid DNA-cationic lipid complexes can betoxic to cells. The SPLP-CPL₄ contain low levels of cationic lipid andare potentially less toxic than complexes. The toxicity of SPLP-CPL₄ andcomplexes was assayed by determining cell viability following a 48 hexposure to levels of SPLP-CPL₄ and complexes corresponding to 0.5 μgplasmid and ˜30 nmol total lipid. As shown in FIG. 37B, SPLP-CPL₄exhibit little if any toxicity. Cell survival was only 30% after a 48 hincubation with Lipofectin complexes whereas ˜95% of the cells wereviable following a 48 hour incubation with SPLP-CPL₄.

[0369] The efficiency of transfection, indicated by the proportion ofcells transfected by a vector, is also an important parameter. Theproportion of cells transfected were estimated using plasmid carryingthe green fluorescent protein (GFP) gene. Transfection was detected byexpression of the fluorescent protein inside a cell employingfluorescence microscopy. As shown in FIGS. 37A and 38B, approximately35% of the cells at 24 h and 50% at 48 h were transfected by SPLP-CPL₄,with no apparent cell death. In contrast, Lipofectin complexes exhibitmaximum transfection efficiencies of less than 35% and only ˜50% cellsurvival after the 24 h transfection period. Similar low transfectionefficiencies and high toxicities were also seen with DOPE:DODACcomplexes.

[0370] The results of this study demonstrate that the incorporation ofCPL₄ into SPLP results in improved uptake into BHK cells and adramatically enhanced transfection potency of SPLP when Ca is present.There are three points of interest. The first concerns the chemicalcomposition and structure of the SPLP-CPL₄ system and the generality ofthe post-insertion procedure for modifying the trophism and transfectionpotency of SPLP. The second concerns the relation between enhanceduptake of SPLP, the presence of Ca²⁺ and the transfection activitiesobserved. Finally, it is of interest to compare the properties of theSPLP-CPL₄ system with plasmid DNA-cationic lipid complexes.

[0371] The second point of discussion concerns the mechanism wherebyCPL₄ increases the transfection potency of the SPLP system. Clearly thepresence of the CPL₄ increases the uptake of SPLP into the BHK cells,however the increase in transfection potency is almost entirelydependent on the additional presence of Ca²⁺. It may be noted that,following an 8 h incubation, the presence of 4 mol % CPL₄ increases theuptake of SPLP into BHK cells by approximately 50-fold, whereas thetransfection potency (in the presence of Ca²⁺) is increased by a factorof ˜10⁴. Previous work conducted on SPLP has shown that the presence ofCa²⁺ results in a maximum increase in transfection potency of ˜600 andthat this increase in potency results from an ability of Ca²⁺ to assistin destabilizing the endosomal membrane following uptake, rather than anincrease in uptake itself. In turn, this suggests that the improvementin transfection potency for the SPLP-CPL₄ system over the SPLP systemarises from the CPL₄-dependent increase in uptake multiplied by theCa²⁺-dependent improvement in intracellular delivery following uptake.

[0372] The final area of discussion concerns the advantages of theSPLP-CPL₄ system over other non-viral vectors, which include thewell-defined modular nature of the SPLP-CPL₄ system as well as toxicityand potency issues. First, the well-characterized nature of theSPLP-CPL₄ as small, homogeneous, stable systems containing one plasmidper particle contrast with non-viral systems such as plasmidDNA-cationic lipid complexes which are large, inhomogeneous, unstablesystems containing ill-defined numbers of plasmids per complex. Animportant point is that SPLP are basic components of more sophisticatedsystems, such as SPLP-CPL₄, which can be constructed in a modularfashion. For example, post-insertion of PEG-lipids which containspecific targeting ligands in place of the cationic groups of CPL shouldresult in SPLP that are specifically targeted to particular cells andtissues. With regard to toxicity, it is clear that SPLP-CPL₄ aremarkedly less toxic to BHK cells in tissue culture. This is presumablyrelated to the low proportions of cationic lipid contained in SPLP ascompared to complexes. The transfection potency and efficiency ofSPLP-CPL₄ is clearly comparable to the levels that can be achieved withcomplexes. It should be noted that this finding suggests that models oftransfection by complexes that involve.

[0373] In the present example, the superiority of SPLP-CPL₄ compared tocommercially available complex systems (e.g. Lipofectin) has beendemonstrated. Thus, a synthetic virus has been developed that will havehigh transfection potency but none of the problems associated withviruses. Many points can be made to corroborate these statements. Thefirst point revolves around the placement of the charge. Whereas oncomplexes the charges are located on the surface of the lipid bilayer,the SPLP-CPL₄ possess charges on the vesicle surface which are localizeda good distance from the liposomal surface, above the protective PEGcoating which surrounds the liposome. In the case of the complexes,proteins binding to the liposome surface can lead to recognition andclearance by macrophages of the RES. (see, Chonn et al., J Biol Chem;267:18759-18765 (1992)) In the SPLP-CPL₄, the charge on the surface ofthe bilayer is protected by the PEG coating, such that this should notoccur. However, the charge on the SPLP-CPL₄ will allow the associationof the liposomes with cells resulting in eventual uptake andtransfection.

[0374] The size and serum stability of the SPLP-CPL₄ compared tocomplexes are important parameters for effective gene delivery systems,especially if one wishes to approach the capabilities of viral systems.The SPLP-CPL₄ have been shown here to be of relative small size (˜100nm) compared to complexes, which are frequently on the order of micronsin diameter. The small size should allow for accumulation at sites withlarger fenestration (e.g. tumors, and inflammation sites). (see, Kohn etal., Lab Invest; 67:596-607 (1992)). As stated earlier, DNA in theSPLP-CPL₄ was shown to be protected from the external environment (i.e.inaccessible to degradation by DNase within serum), whereas DNA incomplexes is susceptible to DNase. (see, Wheeler et al., Gene Therapy;6:271-281 (1999)).

[0375] Viruses (see, Hermonat et al., Proc. Natl. Acad. Sci. USA;81:6466-6470 (1984); Lebkowski et al., Molec Cell Biol; 8:3988-3996(1988); Keir et al., J Neurovirology, 3:322-330 (1997)]and lipid/DNAcomplexes (see, Felgner et al., Proc Natl Acad Sci USA, 84:7413-7417(1987); Felgner et al., J Biol Chem; 269:2550-61 (1994); Hofland et al.,Proc Natl Acad Sci USA; 93:7305-7309 (1996); Bebok et al., J Pharm ExpTher; 279:1462-1469 (1996); Gao et al., Gene Therapy; 2:710-722 (1995))have been shown to possess high in vitro transfection potencies. Ittherefore reasons that the SPLP-CPL₄ system, if it is to attain viralqualities, should be capable of attaining these high transfections. Thishas actually been achieved by the SPLP-CPL₄ system on BHK cells, withtransfection levels reaching a factor of two higher than a commerciallyavailable complex system (i.e. Lipofectin). This is a huge improvementover SPLP, which showed only a small amount of transfection.

[0376] Efficient systemic delivery and transfection of genetic drugs areachieved using this SPLP-CPL₄ system due to the above benefits. Veryhigh transfections in vitro with SPLP-CPL₄ have been achieved. Inaddition, a system wherein the positioning of the positive charges onthe CPL, so that the PEG of the PEG-Cer initially masks it. This isachieved by the synthesis of DSPE-PEG-CPL₄ with a shorter PEG moiety.This allows for its accumulation at disease sites followed by thecontrolled release of the PEG-Cer, exposing the positive charges to thesurrounding cells.

Example X

[0377] This example shows transfection rates of BHK cells by long-versus short-chained CPLs.

[0378] Using synthesis methods from above, CPL (PEG 3.4k) and CPL(PEG1k) were generated and each inserted into a separate SPLP systemcontaining PEG-₂₀₀₀-Cer C20 as described above. FIG. 38 illustratestransfection rates of the CPLs having a PEG 3.4k versus a CPL having aPEG 1k. The short-chained PEG in the CPL results in a decrease by afactor of about 4 compared to the transfection by the long chained CPL.Without being bound by any particular theory, it is believed that thelong chain CPL (PEG₃₄₀₀) sticks out above the surface, whereas the shortchain CPL (PEG₁₀₀₀) is buried (masked) in the surface of the SPLP. Thereduced in vitro transfection of the short chain CPL clearly suggeststhat it has improved in vivo circulation.

Example XI

[0379] This example shows that CPL8 behaves similar to CPL4 with respectto insertion into LUVs, and that transfection can be achieved withCPL8-LUV systems. TABLE 10 Insertion of CPL₈ in SPLP and LUV. Initialmol % CPL₈ % Insertion Final mol % CPL₈ SPLP 1.11 97% 1.07 1.39 85% 1.191.67 95% 1.60 1.94 87% 1.70 LUV 1.05 79% 0.82 1.39 71% 0.99 1.74 76%1.32 2.11 89% 1.88

[0380] The insertions of the CPL₈ into LUV and SPLP is very similar towhat was observed for the insertions of CPL₄. For the transfection anduptake of these particles on BHK cells, variable results are obtained,with the CPL₈ performing better than the CPL₄ sometimes and vice versaat other times.

Example XII

[0381] In this in vitro example using mouse neuroblastoma cell lineNeuro-2a (ATCC—CCL-131), the SPLP-CPL4[1k] is used to determine geneexpression with respect to varying Ca²⁺ concentrations and to compare togene expression using a standard SPLP (PEG-CerC20 10%; CPL₄[1k] 4%; andother components; DNA:lipid ratio=0.05).

[0382] 5×10⁴ cells/well are plated in 24-well plates in 1 mL of completemedia (MEM(Eagle) with non-essential amino acids and Hanks' bufferedsalt solution with 10% FBS). Plates are incubated overnight at 37° C.with 5.0% CO₂. To each group set out below is added 500 μL transfectionmedia in triplicate. TABLE 11 SPLP or SPLP-CPL [Ca2+] Complete GROUP(μg) (250 mM) (μL) Media (μL) A (0 mM Ca2+) 2.5 0 1980 B (2 mM Ca2+) 2.548 1932 C (4 mM Ca2+) 2.5 96 1884 D (6 mM Ca2+) 2.5 144 1836 E (8 mMCa2+) 2.5 192 1788 F (10 mM Ca2+) 2.5 240 1740 G (12 mM Ca2+) 2.5 2881692 H (14 mM Ca2+) 2.5 336 1644

[0383] 2.5 μg DNA is added per well in fully encapsulated SPLPs (0.5 mLtotal solution). Plates are incubated for 8 hrs. Transfection media isremoved. 1 mL of complete media is added back. Cells are incubate foranother 24 hrs at 37° C., 5.0% CO₂.

[0384] For analysis, media is removed from cells and they are washed 2×with PBS then frozen at −70° C. Cells are lysed with 150-200 μL 1×CCLR;then shaken 5 minutes on plate shaker. 20 μL lysate is transferred to a96-well luminescence plate. Plates are read to determine luciferaseactivity.

[0385] The results are shown in FIG. 39. As shown therein, SPLP+4 mol %CPL4-1k produces 4 orders of magnitude of gene expression more than SPLPalone in Neuro-2a cells. Effects of calcium are not considered to besignificant in this experiment. The amount of luciferase producedremains the same from 2-14 mM Ca2+.

Example XIII

[0386] This in vivo example discloses pharmacokinetics andbiodistribution of CPL₄-1-k LUVs (SPLPs containing short chain CPLs) inC57/b16 mice. Different SPLP formulations containing increasing amountsof CPL-4-1k are assayed in vivo to determine optimal clearancecharacteristics.

[0387] CPL₄-1k SPLPs are prepared according to previous protocols.Before use, all samples are characterized to determine actualcomposition prior to administration. All samples are filter sterilizedprior to dilution to working concentration. All samples are to providedin sterile crimp top vials. All vials are labeled with the formulationdate, lipid composition, and specific activity. ³[H]CHE is incorporatedat 1 μCi/mg Lipid. The following formulations are made and analyzed:

[0388] A: ³[H]CHE-LUV DOPE:DODAC:PEGC20:84:6:10

[0389] B: ³[H]CHE-LUV DOPE:DODAC:PEGC20:84:6:10+1 mol % CPL-4-1k

[0390] C: ³[H]CHE-LUV DOPE:DODAC:PEGC20:84:6:10+2 mol % CPL-4-1k

[0391] D: ³[H]CHE-LUV DOPE:DODAC:PEGC20:84:6:10+3 mol % CPL-4-1k

[0392] E: ³[H]CHE-LUV DOPE:DODAC:PEGC20:84:6:10+4 mol % CPL-4-1k

[0393] Experiments used 100 C57/b16 mice, female, 18-23 g all orderedfrom Harlan Sprague Dawley. All animals housed in cages of 4 animals pergroup in 25 groups. TABLE 12 Group Mice Treatment Timepoint Assay A 4A:DOPE:DODAC:PEGC20:84:6:10 15 min PK B 4 A:DOPE:DODAC:PEGC20::84:6:10 1 hr PK C 4 A:DOPE:DODAC:PEGC20::84:6:10  4 hr PK D 4A:DOPE:DODAC:PEGC20::84:6:10  8 hr PK E 4 A:DOPE:DODAC:PEGC20::84:6:1024 hr PK F 4 B:DOPE:DODAC:PEGC20::84:6:10 + 1 mol % CPL-4-1k 15 min PK G4 B:DOPE:DODAC:PEGC20::84:6:10 + 1 mol % CPL-4-1k  1 hr PK H 4B:DOPE:DODAC:PEGC20::84:6:10 + 1 mol % CPL-4-1k  4 hr PK I 4B:DOPE:DODAC:PEGC20::84:6:10 + 1 mol % CPL-4-1k  8 hr PK J 4B:DOPE:DODAC:PEGC20::84:6:10 + 1 mol % CPL-4-1k 24 hr PK K 4C:DOPE:DODAC:PEGC20::84:6:10 + 2 mol % CPL-4-1k 15 min PK L 4C:DOPE:DODAC:PEGC20::84:6:10 + 2 mol % CPL-4-1k  1 hr PK M 4C:DOPE:DODAC:PEGC20::84:6:10 + 2 mol % CPL-4-1k  4 hr PK N 4C:DOPE:DODAC:PEGC20::84:6:10 + 2 mol % CPL-4-1k  8 hr PK O 4C:DOPE:DODAC:PEGC20::84:6:10 + 2 mol % CPL-4-1k 24 hr PK P 4D:DOPE:DODAC:PEGC20::84:6:10 + 3 mol % CPL-4-1k 15 min PK Q 4D:DOPE:DODAC:PEGC20::84:6:10 + 3 mol % CPL-4-1k  1 hr PK R 4D:DOPE:DODAC:PEGC20::84:6:10 + 3 mol % CPL-4-1k  4 hr PK S 4D:DOPE:DODAC:PEGC20::84:6:10 + 3 mol % CPL-4-1k  8 hr PK T 4D:DOPE:DODAC:PEGC20::84:6:10 + 3 mol % CPL-4-1k 24 hr PK U 4E:DOPE:DODAC:PEGC20::84:6:10 + 4 mol % CPL-4-1k 15 min PK V 4E:DOPE:DODAC:PEGC20::84:6:10 + 4 mol % CPL-4-1k  1 hr PK W 4E:DOPE:DODAC:PEGC20::84:6:10 + 4 mol % CPL-4-1k  4 hr PK X 4E:DOPE:DODAC:PEGC20::84:6:10 + 4 mol % CPL-4-1k  8 hr PK Y 4E:DOPE:DODAC:PEGC20::84:6:10 + 4 mol % CPL-4-1k 24 hr PK

[0394] Mice were treated with ³[H]CHE-LUV administered by tail vein I.V.in a total volume of 200 μl . Mice receive one treatment only. At theindicated time-points mice are weighed, sacrificed, and blood will becollected by cardiac puncture then evaluated for ³[H]CHE. Formulationsare expected to be well tolerated. Mice are treated according tocertified animal care protocols. Any mice exhibiting signs of distressassociated with the treatment are terminated at the discretion ofvivarium staff. All mice are terminated by CO₂ inhalation followed bycervical dislocation. Measurement of ³[H]CHE from blood is determinedaccording to standard protocols.

[0395] In vivo pharmacokinetics of SPLP containing short chain CPL₄ areillustrated in FIG. 40. It is observed that that increasing amounts ofthe CPL₄ in the SPLP tends to increase the rate of clearance from theblood. CPL₄ incorporated a 1 mol % gives clearance results which aresimilar to SPLPs without CPL₄. Incorporation of higher amounts of CPL4tends to increase the rate of clearance of the SPLP from the blood.SPLP-CPL₄ [1k] (1%) shows best plasma clearance characteristics with at_(1/2) of 6-7 hours. Anything greater than 1 mol % clears more rapidly.

[0396] The results disclosed in this specification indicate a furtherrefinement of SPLP technology. In particular, from these results it isclear that the type of CPL (i.e. the length of the polymer chain; andthe amount of cationic charge per molecule) and the amount of such CPLin an SPLP must be optimized to obtain the best balancing of clearanceproperties in vivo with enhanced transfection ability. In vitro data hasshown long chain CPLs and higher levels of such CPLs are to be preferredto increase transfection. However, as seen in previous comparisons ofSPLPs versus lipid complexes, lipid formulations that work best in vitroare not best suited in vivo. In vivo results herein demonstrate thatshort chain CPLs incorporated at approximately 1% are optimized forcirculation lifetimes in animals.

[0397] It is understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and scope of the appended claims. All publications, patents,and patent applications cited herein are hereby incorporated byreference in their entirety for all purposes.

1. A compound having the general structure of Formula I: A-W-Y  Iwherein: A is a lipid moiety; W is a hydrophilic polymer; and Y is apolycationic moiety.
 2. The compound according to claim 1, wherein saidhydrophilic polymer is non-immunogenic or weakly immunogenic.
 3. Thecompound according to claim 1, wherein W is a polymer selected from thegroup consisting of PEG, polyamide, polylactic acid, polyglycolic acid,polylactic acid/polyglycolic acid copolymers and combinations thereof,said polymer having a molecular weight of about 250 to about 7000daltons.
 4. The compound according to claim 1, wherein Y comprises aleast one basic amino acid or derivative thereof.
 5. The compoundaccording to claim 1, wherein Y has at least 4 positive charges at aselected pH.
 6. The compound according to claim 1, wherein Y has atleast 8 positive charges at a selected pH.
 7. The compound according toclaim 4, wherein Y comprises an amino acid selected from the groupconsisting of lysine, arginine, asparagine, glutamine, derivativesthereof and combinations thereof.
 8. The compound according to claim 2,wherein A is a member selected from the group consisting of adiacylglycerolyl moiety, a dialkylglycerolyl moiety, a N—N-dialkylaminomoiety, a 1,2-diacyloxy-3-aminopropane moiety and a1,2,dialkyl-3-aminopropane moiey.
 9. The compound according to claim 3,wherein W is PEG.
 10. The compound according to claim 3, wherein W is apolyamide polymer.
 11. The compound according to claim 3, wherein W hasa molecular weight of about 250 to about 2000 daltons.
 12. The compoundaccording to claim 9, having the general structure of Formula II:

wherein X is a member selected from the group consisting of a singlebond and a functional group covalently attaching said lipid moiety A toat least one ethylene oxide unit; Z is a member selected from the groupconsisting of a single bond and a functional group covalently attachingat least one ethylene oxide unit to the polycationic moiety Y; and n isan integer having a value of between about 6 and about
 50. 13. Thecompound according to claim 12, wherein X is a member selected from thegroup consisting of a single bond, phosphatidylethanolamino,phosphatidylethanolamido, phosphoro, phospho, phosphoethanolamino,phosphoethanolamido, carbonyl, carbamate, carboxyl, carbonate, amido,thioamido, oxygen, sulfur and NR, wherein R is a hydrogen or alkylgroup.
 14. The compound according to claim 12, wherein Z is a memberselected from the group consisting of a single bond,phosphatidylethanolamino, phosphatidylethanolamido, phosphoro, phospho,phosphoethanolamino, phosphoethanolamido, carbonyl, carbamate, carboxyl,carbonate, amido, thioamido, oxygen, sulfur and NR, wherein R is ahydrogen or alkyl group.
 15. The compound according to claim 12, whereinA is a diacylglycerolyl moiety; X is phosphoethanolamido; Z is NR,wherein R is a hydrogen atom; and Y comprises about 1 to about 10 basicamino acids or derivatives thereof.
 16. The compound according to claim15, where A is a diacylglycerolyl moiety having 2 fatty acyl chains,wherein each acyl chain is independently between 2 and 30 carbons inlength and is either saturated or has vary degrees of saturation. 17.The compound according to claim 15, wherein Y comprises an amino acidselected from the group consisting of lysine, arginine, glutamine,derivatives thereof and combinations thereof.
 18. The compound accordingto claim 15, wherein A is a diacylglycerolyl moiety having 2 fatty acylchains, wherein each acyl chain is a saturated C-18 carbon chain; and Yis a cationic group comprising 4 lysine residues or derivatives thereof.19-54. (canceled)